Therapeutic viral microparticles for promoting stent biofunctionality and wound healing in vertebrate individuals

ABSTRACT

The present disclosure provides viral microparticles comprising genetically-engineered baculoviruses (at least partially) embedded in a polymeric matrix for the local delivery of therapeutic nucleic acid molecules to the cells of a vertebrate individual (optionally in combination with a medical implant such as vascular stent platform). The viral microparticles are especially useful for promoting the healing of a wound as well as the repair of a blood vessel and prevent pathological scarring. Also provided herein are processes for making the viral microparticles, pharmaceutical compositions comprising viral microparticles as well as supports comprising the viral microparticles for the locating the viral microparticles in a wound or in the vicinity of a wound.

CROSS-REFERENCE TO RELATED APPLICATIONS AND DOCUMENTS

This application claims priority from U.S. provisional application61/811,203 filed on Apr. 12, 2013. The content of this priorityapplication is incorporated herewith in its entirety.

This application also contains a sequence listing in electronic format.The content of this sequence listing is incorporated herein in itsentirety.

TECHNOLOGICAL FIELD

The present disclosure relates to the specific and local delivery oftherapeutic nucleic acid molecules to the cells of a vertebrate host forpromoting the repair of damaged blood vessels and also for wound healingapplications. The therapeutic nucleic acid molecules are provided bypolymeric viral microparticles allowing the controlled-release ofgenetically-engineered baculovirus. The viral microparticles mayoptionally be formulated in pharmaceutical compositions and/or includedin a support for locating at the site of the wound.

BACKGROUND

Percutaneous transvascular coronary angioplasty and stenting is one ofthe most commonly employed interventional procedures for the treatmentof coronary artery disease. A frequent long-term complication of thistreatment modality is the phenomenon of in stent restenosis (ISR) whichoccurs at the site of the atherosclerotic lesion, leading to theobstruction of dilated arteries in 20-30% of patients within 6 months ofstenting. The primary contributors of this multifactorial pathologicalevent are incomplete endothelial recovery and vascular SMC proliferationin the inner lining of the artery. Several approaches have been used toimprove stent design and durability, such as the use of covered stentsto improve biocompatibility of the stent material and intracoronaryradiation to inhibit inflammation and proliferation of smooth musclecells. Radiation therapy, although effective, was accompanied by delayedhealing and incomplete endothelial recovery; whereas coated stents havenot been completely successful in eliminating the problem.

The introduction of drug eluting stent (DES) has been seen as asignificant improvement in the existing stent design. The drugs used aremostly antiproliferative which target the smooth muscle cellproliferation and related inflammatory systems. But the direct use ofthese chemical factors is limited by the problems associated with drugwashout, their inadvertent effects on non-target cells, as well as theunselected inhibition of vascular cell division by the drugs leading toincomplete endothelial recovery of stented vessels. Moreover, recentlong term meta-analysis of DES studies demonstrating increased risk oflate-stent thrombosis has raised questions on the long term safety ofDES. As the pathological recurrence of stenosis mainly stem from theendothelial cell lining damage and dysfunction caused by the stentimplantation, approaches to promote accelerated re-endothelializationcan be of significant importance to reduce the risk of ISR and latein-stent thrombosis. Moreover, this strategy will evade the harmfulconsequences of discontinuation of antiplatelet drug therapy which areotherwise needed post DES implantation. Therapeutic induction ofvascular cells by transferring proangiogenic vascular genes, such asVegf, to promote re-endothelization has been proposed as a potent way toattenuate neointima formation and reduce restenosis. Vegf cytokines area family of endothelial specific mitogenic factors that bind to tyrosinekinase receptors 1 and 2, expressed almost exclusively on theendothelial cells, and widely used for selective proliferation andrepair of damaged vascular endothelial structures. Although mammalianviral vectors have been efficiently used for vascular gene therapy,clinical applications are limited due to safety concerns related to riskof inclusion of replication competent viruses and proper optimization ofthe human tolerization level to these pathogens. Moreover, there areassociated problems of immunogenicity due to high viral titer, inductionof innate toxicity and inflammatory reactions coupled with the inherentrisk of viral integration to the host genome. On the other hand,non-viral gene delivery systems are mainly limited by low in vivo genetransfer efficiencies.

Wound healing is a complex physiological event which leads to thereplacement of the injury tissue by a new one. The adequate healing of awound requires the proliferation and/or dedifferentiation of cells inthe vicinity of the wound as well as the production and/or remodeling ofthe extracellular matrix, but also a return to quiescence once the scarhas been formed (differentiation of cells, halt in proliferation and inmatrix remodeling). In some pathological instances, the healing processfails or delays the return to quiescence and as such promotes thehyperplasia of cells, the production of an abnormal amount ofextracellular matrix (e.g. too much or too little) and can even lead tothe formation of a pathological scar (e.g. hypotrophic/hypertrophic scarin the skin, restenosis in large vessels for example).

In situations where the wound healing process is susceptible to inducepathological side-effects (in stent restenosis for example), it is wouldbe desirable to be provided with a gene-delivery system which couldfavor the homeostatic wound healing process by providing, locally, inthe vicinity of a wound, a therapeutic nucleic acid molecule fortransduction by the cells of the host. Since the process of woundhealing is performed within a specific time window, in some embodiments,it would be highly desirable to be provided with a gene-delivery systemwhich allows a transient transduction of the therapeutic nucleic acidmolecule during to the wound healing process. In addition, since a woundis usually limited to a specific location within the host, in someembodiment, it would be highly desirable to be provided with agene-delivery system which specifically allows the transduction of thetherapeutic nucleic acid to the vicinity of the wound. Preferably, thegene-delivery system limits (and in some embodiments) prevents theimmune reaction associated with the transduction of the therapeuticnucleic acid molecule.

BRIEF SUMMARY

The present disclosure provides viral microparticles in which embeddedgenetically-engineered baculoviruses are used to deliver a therapeuticnucleic acid molecule to the cells of the vertebrate individual. Theviral microparticles can be formulated in a pharmaceutical compositionand optionally be included in a support. The viral microparticles favoradequate repair of blood vessels as well as wound healing.

According to a first aspect, the present disclosure provides a viralmicroparticle for the delivery of a recombinant therapeutic nucleic acidmolecule to the cells of a vertebrate individual. The viralmicroparticle comprises, as a first component, a matrix of biocompatiblebiodegradable polymers. The viral microparticle further comprises, as asecond component, at least one (or in an embodiment a plurality of)genetically-engineered baculovirus having a viral genomecomprising/encoding the recombinant therapeutic nucleic acid moleculeand lacking the ability of replicating in the cells of the vertebrateindividual. In an embodiment, the biocompatible biodegradable polymeris/comprises a polyester. In another embodiment, the polyesteris/comprises poly(lactic-co-glycolic acid). In still another embodiment,the genetically-engineered baculovirus is from the generanucleopolyhedrovirus, and in still a further embodiment, thegenetically-engineered baculovirus is a multicapsid virus. In yet afurther embodiment, the genetically-engineered baculovirus is from thesubtype Autographa californica multicapsid nucleopolyhedrovirus(AcMNPV). In an embodiment, the genetically-engineered baculovirus is atleast partially embedded in the matrix. In another embodiment, therecombinant nucleic acid molecule encodes a growth factor, such as, forexample, an angiogenic growth factor (e.g. vascular endothelial growthfactor (VEGF)). In another embodiment, the genetically-engineeredbaculovirus further comprises a coat of cationic polymers covering atleast a portion of the surface of the baculovirus. In a furtherembodiment, the cationic polymers are/comprise dendrimers. In still afurther embodiment, the dendrimers are/comprise poly(amidoamine)(PAMAM), such as, for example, a G0 PAMAM. In an embodiment, the viralmicroparticle has a relative diameter between about 5 μm and about 10μm.

In a second aspect, the present disclosure provides a process for makinga viral microparticle for the delivery of a recombinant therapeuticnucleic acid molecule to the cells of a vertebrate individual. Broadly,the process comprises (a) resuspending, in an aqueous solution, agenetically-modified baculovirus having a viral genome comprising therecombinant therapeutic nucleic acid molecule so as to obtain an aqueouspreparation; (b) combining and homogenizing the aqueous preparation ofstep (a) with a solution of a biodegradable biocompatible polymer and awater immiscible, volatile organic solvent so as to form a water-in-oil(w/o) emulsion; (c) combining and stirring (and optionally homogenizing)the water-in-oil emulsion of step (b) with an oil so as to form awater-in-oil-in-oil (w/o/o) emulsion; and (d) evaporating the waterimmiscible, volatile organic solvent from the water-in-oil-in-oilemulsion of step (d) so as to form the viral microparticles. In anembodiment, the process further comprises (e) recuperating and washingthe viral microparticles obtained in step (d). In an embodiment, the oilfurther comprises a surfactant, such as, for example, a non-ionicsurfactant. In another embodiment, the oil is a vegetable oil. In yetanother embodiment, the process further comprises coating, at leastpartially, the genetically-modified with a cationic polymer prior tostep (a).

In a third aspect, the present disclosure provides a viral microparticlefor the delivery of a recombinant therapeutic nucleic acid molecule to avertebrate individual obtained by the process described herein.

In a fourth aspect, the present disclosure provides a pharmaceuticalcomposition having (i) a gelling agent and (ii) the viral describedherein or obtained by the process described herein. In an embodiment,the gelling agent comprises a protein and/or a protein fragment, suchas, for example, fibrinogen and/or fibrin.

In a fifth aspect, the present disclosure provides a process forformulating the pharmaceutical composition described herein. Broadly,the process comprises combining the gelling agent and the viralmicroparticle. In an embodiment, the process further comprisescross-linking the gelling agent.

In a sixth aspect, the present disclosure provides a kit for making thepharmaceutical composition described herein. The kit comprises thegelling agent described herein and the viral microparticle describedherein In an embodiment, the kit can further comprise a cross-linkingagent.

In a seventh aspect, the present disclosure provides a supportcomprising the viral microparticle described herein or obtained by theprocess described herein. Alternatively, the support comprises thepharmaceutical composition described herein or made by the processdescribed herein. In an embodiment, the support is solid. In anotherembodiment, the support is metallic. In still another embodiment, thesupport is a medical implant, such as, for example a stent.

In an eighth aspect, the present disclosure provides a method ofpromoting the healing of a wound in a vertebrate individual in needthereof. The method comprising placing a therapeutic effective amount ofat least one of: the viral microparticle described herein, the viralmicroparticle obtained by the process described herein, thepharmaceutical composition described herein, the pharmaceuticalcomposition obtained by the process described herein, or the supportdescribed, in the vicinity of the wound so as to favor the healing ofthe wound in the vertebrate individual. Also contemplated herein theviral microparticle described herein, the viral microparticle obtainedby the process described herein, the pharmaceutical compositiondescribed herein, the pharmaceutical composition obtained by the processdescribed herein, or the support described herein for promoting thehealing of a wound in a vertebrate individual in need thereof as well asthe use of the viral microparticle described herein, the viralmicroparticle obtained by the process described herein, thepharmaceutical composition described herein, the pharmaceuticalcomposition obtained by the process described herein, or the supportdescribed herein for promoting the healing of a wound in a vertebrateindividual in need thereof

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the nature of the disclosure, referencewill now be made to the accompanying drawings, showing by way ofillustration, a preferred embodiment thereof, and in which:

FIG. 1 provides a schematic representation of design, formulation andmode of action of virus loaded bioactive stent to attenuate ISR. (A)Release of entrapped viruses carrying an exemplary therapeutic nucleicacid molecule encoding angiogenic transgene (Vegf) to the adheringvascular wall. (B) Local overexpression of transgene to enhanceendothelial regeneration. (C) Proper cushioning of intima layer withregenerated endothelial monolayer to reduce risk of restenosis by smoothmuscle proliferation and thrombosis by avoiding further exposure toblood. With time, the stent loses its coatings due to the biodegradablenature of the polymers, leaving behind the bare stent struts in thealready recovered vascular segment. (D) Schematic representation ofgeneration of Bac-PAMAM complex and its subsequent microencapsulation inPLGA microspheres by a w/o/o double emulsion solvent evaporation method.

FIG. 2 illustrates SEM (A and A′) and AFM (B and B′) microphotographs ofvirus loaded PLGA microspheres demonstrating the surface morphologyprepared by an embodiment of the method described herein (andillustrated in FIG. 1D). TEM picture of empty microspheres (C) and virusloaded microspheres (D). (E) represents the ultrathin innercross-sections of the microspheres loaded with viruses under TEM. Note:The entrapped viruses have a circular shape in the TEM picture becausethe microphotograph was taken for a single plane on the cutcross-section of the virus loaded microsphere.

FIG. 3 illustrates the characterization of PLGA MS coated fibrin stent.(A) Uncoated Bare metal stent, (B) Nile red containing MS loaded stentafter coating and (C) expanded stent after crimping on a ballooncatheter. (D) Fluorescence image of the stent showing the fluorescentPLGA MS embedded on the fibrin stent surface. SEM microphotographs of(E) bare metal stent, (F) fibrin coated stent and (G i and ii) PLGA MSloaded fibrin coated stents. The image in (G ii) is a magnified image ofthe inset of (G i). (H) Cumulative baculovirus release (in terms of % ofinitial virus loaded) from stents coated with varying concentrations (50and 100 mg/ml DCM) of PLGA MS prepared by w/o/w and w/o/o method showscontrolled release of virus load over 24 h as a function of the PLGAconcentration and MS preparation method. Results are shown as thepercentage of active virus cumulative release in function of hours postincubation. Results are shown for MS baculovirus (50) obtained by awater-in-oil-in-oil process (♦) or a water-in-oil-in-water process (▪)as well as for MS baculovirus (100) obtained by a water-in-oil-in-oilprocess (▴) or a water-in-oil-in-water process (X).

FIG. 4 illustrates the surface modification of baculovirus with PAMAMdendrimer (G0). Zeta potential (in mV) (A) and particle size (in nM) (B)of Bac-PAMAM (0) (free Bac), Bac-PAMAM (0.01), Bac-PAMAM (0.1),Bac-PAMAM (0.5), and Bac-PAMAM (1.0), where the values within bracketsindicate the ratio of PAMAM molecules in μmol per 10⁸ viruses. The datais represented by mean±standard deviation (SD). TEM images of free Bac(C) and Bac-PAMAM (0.5) (D) suspended in PBS. Arrows indicate thepositively charged PAMAM dendrimer coating on the negatively chargedbaculovirus surfaces to form the hybridized nanostructures. Scaleindicates 100 nm length.

FIG. 5 illustrates the characterization of PLGA-encapsulated Bac. (A)The kinetics of PLGA-encapsulated BacMGFP with or without PAMAMcoatings. The prepared stents with encapsulated viral preparationscontaining 0, 0.01, 0.1, 0.5 and 1.0 μmol PAMAM molecules per 10⁸baculovirus were incubated in PBS. At the indicated times PBS wascollected and used for transduction of HASMCs using standard procedurementioned in the Example section. Results are shown as the percentage oftransduced cells for each type of encapsulated baculovirus 12 h, 18 h or24 h after transduction. Data represent mean±SD (n=3). (B-E) representsthe transduced cells 24 h post transduction under fluorescence fieldwhile (B′-E′) represents the corresponding bright field. (F) Cytotoxiceffect of the above encapsulations on HASMCs after 12 h of incubationwas analyzed. Data from different groups were represented in terms ofpercentage of viable cells in function of the type of encapsulatedbaculovirus. **=P<0.01 compared to control Bac group (n=3).

FIG. 6 illustrates the effect of serum and storage temperatures onbioactivity of BacMGFP loaded stent. The BacMGFP-PAMAM MS, BacMGFP MSand BacMGFP stents, after 1 h incubation in 50% serum or PBS solution,were used to transduce 2×10⁴ HASMCs per well in 96 well-plate usingstandard method as mentioned in Example I. Similarly, BacMGFP-PAMAM MSstents after storage for 3 months at different temperatures (4° C., −20°C., −80° C. and control freshly prepared) were used to transduce HASMCsin vitro. Data was represented in terms of normalized MGFP expressiontaking BacMGFP-PAMAM MS expression as 100% (A) and taking freshlyprepared stent expression as 100% (B). Data represent mean±SD (n=3).Statistically significant differences within groups are denoted by***=P<0.001 and **=P<0.01, while condition-matched differences betweengroups are represented by y<0.001 (PBS) and #<0.001 (serum).

FIG. 7 illustrates the release and expression of a therapeutic nucleicmolecule from the encapsulated baculoviruses. (A) Quantification of VEGFreleased by transduced HASMCs over time as detected by ELISA. Resultsare shown as released VEGF (ng/mL) in function of days post-transductionfor various types of baculovirus (Bac-PAMAM=♦, Bac only=▪ andcontrol=▴). (B) Proliferation of HUVECs grown in the presence ofconditioned medium (CM from Day 4) from BacVegf-PAMAM (with or withoutanit-VEGF Ab) and BacVegf transduced HASMCsAs control group, CM fromnon-transduced cells (Ctrl) was taken. Initial seeding density was 2×10⁴cells per well in 96 well plate and cell proliferation was detected by acolorimetric assay on day 3. Results are shown for the Day 4 conditionedmedium obtained from different types of baculovirus (Control, Bac Vegfonly, BacVegf-PAMAM (0.5) and Bac Vegf-PAMAM with anti-VEGF antibodies).ANOVA: Significantly higher values in groups compared to control aredenoted by ***=P<0.001 and **=P<0.01. (C) Induction of HUVEC migrationin an in vitro wound healing assay. Results are shown as the percentageof would healing in function of different types of encapsulatedbaculoviruses or control (control, Bac Vegf only, Bac Vegf-PAMAM or BacVefg-PAMAM with anti-VEGF antibodes). HUVEC cell monolayer was woundedwith cell scraper and treated with CM from above mentioned groups. HUVECwere photographed (200×) after 24 h treatment and percentage ofscratched area (marked by the white dotted border line) covered by themigrated cells were analyzed using Image J software (C i=representativeimage of the control, ii=representative image of Bac Vegf only,iii=representative image of Bac Vegf-PAMAM and iv=representative imageof Bac Vegf-PAMAM and anti-VEGF antibodies). (D) Effect of CM from abovementioned groups on tubular formation in vitro. After 18 hco-cultivation with HUVECs, tubular formation was evaluated. The graphsrepresent the relative tubule length in μm taking BacVegf as 100% (D i)and counting of capillary tubule number (D ii). The data represent themean±SD of three independent experiments. (D iii-vi) Properly formedtubular structure was observed in BacVegf-PAMAM (v) and BacVegf (iv)groups, compared to the unstimulated control (iii) and Ab treated (vi)group as examined using a fluorescent microscope under 100×magnification.

FIG. 8 illustrates the experimental procedure and results for the invivo experiment in a canine model. The femoral artery was first isolatedfrom the normal blood flow using clips (A i). The selected portion wasthen flushed with PBS followed by severe balloon injury using ballooncatheter (A ii). This was followed by the insertion of the crimpedpolymer coated stent in the damaged artery. The balloon cathetercarrying the stent was inflated to implant the stent at the site. Thiswas followed by deflation and removal of the balloon catheter (A iii).(B) hVegf transgene delivery and expression in the artery. RT-PCR oftissue retrieved from stented vessel segments was performed to identifythe hVegf gene, detecting PCR product. Two (2) weeks post stentimplantation in Coated (+) group, the VEGF transcript was still presentin the proximal (P), middle (M) and distal (D) portions of the stentedartery. But transcript was undetectable in the artery sections 1 cmproximal (P′) and distal (D′) to the stented portion as well as in theCoated (−) control group on both week 2 and week 16. Importantly, thetranscript also disappeared in Coated (+) group by week 16 confirmingthe transgene nature of expression of the viral system. (C)Immunohistochemical localization of VEGF within stented femoral arteryin Coated (+) group at week 2 post stent deployment in the proximal (i),middle (ii) and distal (iii) portions of the stented artery as well asin a control artery (iv). Coated (−) was taken as control. The VEGFexpression was noticed mainly in the intima and outer medial layer. Notethat the expression occurred mainly at the strut area (white dotted)where the stent surface touched the inner lining of the artery, with nosignificant expression in the neointimal area indicating that the genetransfer from stent surface occurred only at the very early stage ofdeployment.

FIG. 9 illustrates the re-endothelialization of vessels following stentimplantation. (A) Evans blue staining confirms that Coated (+) group (i)was able to recover the injury by endothelialization while in Coated (−)group (ii) the wounds was still exposed with high amount of Evans blueuptake. Control artery segment with no injury showed no signs of dyeuptake (iii). (B) Quantification of percentage re-endothelializationweek 2 after staining using imaging software in the Coated (+) andCoated (−) group. The data represent the mean±SD (n=3). (C) SEM picturesof Coated (−) stent on week 2 (C i) and week 16 (C ii) and that ofCoated (+) stents on week 2 (C iii) and week 16 (C iv). Vessels fromCoated (−) group lacked endothelial cell morphology between struts,while endothelial cell monolayer completely covered the stent surfacewith typical fusiform morphology and intact borders in Coated (+) group.(D) Histological assessment of re-endothelialization of arterial tissuesections (measured as the percentage of re-endothelialization at week 16after deployment) in Uncoated, Coated (−) and Coated (+) at week 16 poststent deployment. The data represent the mean±SD (n=8). ANOVA:***=P<0.001 and *=P<0.05, while P value on comparing Coated (+) andCoated (−) in (D) is denoted by y.

FIG. 10 illustrates the effect of BacVegf-PAMAM delivery on ISR assessedby angiography analysis. Comparison of angiography studies at week 16after stent deployment in femoral arteries of dogs. Representativeangiographic images of femoral arteries with uncoated bare metal stent(A′) and stents coated carrying BacNull-PAMAM (B′) and BacVegf-PAMAM(C′) at week 16 after stent deployment, where (A), (B) and (C) shows thecorresponding stent positions before angiography (black arrows). Imageanalysis within the proximal (dotted white arrows) and distal (whitearrows) regions of stented arteries (dotted line) demonstratedsignificantly reduced ISR in Coated (+) group compared to Uncoatedgroup, although significance was not achieved when compared to Coated(−)(D). Results are shown as the percentage of stenosis area as determinedby angiography in function of the different treatment. The datarepresent the mean±SD (n=8). ANOVA: ***=P<0.001. P value on comparingCoated (+) and Coated (−) is denoted by ψ.

FIG. 11 (A i-iii) Cross-sectional view of stented artery through SEMdemonstrating the intimal hyperplasia over the protruded stent struts.(B) The effect of BacVegf-PAMAM delivery on ISR assessed byhistomorphometric analysis. Comparison of histomorphometric studies atweek 16 after stent deployment in dog femoral arteries. Representativecross-sectional images of elastic Van Gieson stained femoral arterieswith uncoated bare metal stent (B i-iii) and stents coated carryingBacNull-PAMAM (C i-iii) and BacVegf-PAMAM (D i-iii) at week 16 afterstent deployment. Percentage stenosis (E) and mean neointimal area (F)analysis at the stented regions demonstrated significantly reduced ISRin Coated (+) group compared to Uncoated and Coated (−) groups. The datarepresent the mean±SD (n=8). ANOVA: **=P<0.01; P value on comparingCoated (+) and Coated (−) is denoted by y.

DETAILED DESCRIPTION

In accordance with the present disclosure, there is provided a viralmicroparticle for promoting wound healing in a vertebrate individual.The viral microparticle is made up of a polymeric matrix in whichgenetically-engineered baculoviruses are at least partially embedded.The viral microparticles are to be located in the vicinity of a woundand are designed to locally discharge or elute, in a controlled fashion,the embedded genetically-engineered baculoviruses. Thegenetically-engineered baculovirus serve as a delivery system tointroduce a therapeutic nucleic acid molecule into the cells in thevicinity of the wound and allow for the transduction of the therapeuticnucleic acid molecule by the cellular machinery of the infected cells.In order to locate the viral microparticles in the vicinity of a wound,it is possible to formulate them as pharmaceutical formulations and eveninclude them onto a support (such as a stent for example). The presentdisclosure also provides methods of using the viral microparticles topromote wound healing in a vertebrate individual.

In embodiments of the present disclosure in which the therapeuticnucleic acid molecule is intended to favor the healing of a wound causedby the insertion of a stent in a large vessel, an approach whereinvertebrate originated insect cell specific recombinant baculovirus canbe used to locally deliver a therapeutic nucleic acid molecule (e.g. aVEGF transgene), thereby promoting the endothelialization of the stentand preventing ISR. Baculovirus offers a unique advantage over otherdelivery systems because of its ability to efficiently transducenon-dividing cells, inherent inability to replicate in mammalian cells,low cytotoxicity even at high viral dosage, absence of preexistingantibodies against baculovirus in animals and ease of production scaleup to high titers. To protect the baculovirus from serum inactivationand achieve a controlled release at the target site, the baculovirus isembedded (at least partially) within polymeric-based microparticles. Insome optional embodiments, it may also be beneficial to enhance the genetransduction efficiency by surface-functionalizing the baculovirus withcationic polymer (such as, for example a synthetic PAMAM dendrimer).Further, in alternative embodiments, to locate the viral microparticleson the stent, a coat of multi-layered fibrin can be used.

Without wishing to be bound to theory, it is postulated that, in anembodiment of the present disclosure, e.g. a therapeutic gene elutingstent formulation, can efficiently deliver angiogenic vascular genes tothe affected site and induce favorable therapeutic effect in the localvascular biology as illustrated in FIG. 1. FIG. 1A shows the initialstage of wound repair by the coated stent, an embodiment of the presentdisclosure. A stent 071 comprising PLGA microspheres 060 with entrappeddendrimer-coated and genetically-engineered baculoviruses 020 is placeda location 070. At location 070, the endothelium that has been damaged(for example by the placement of the stent 071). Location 070 comprisesa smooth muscle cell layer 010 (also referred to as a media) and injuredendothelial cell monolayer 030 (also referred to as an intima). In theembodiment described on FIG. 1A, the stent comprises a fibrin layer 050comprising viral microspheres 060. Viral microspheres 060 are eitheroutside or inside from the fibrin layer 050 and can release thegenetically-engineered viruses 020 in the vicinity of location 070. FIG.1B shows the transduction of the virally-delivered therapeutic nucleicacid molecule by cells located in the vicinity of the wound/stentlocation. Cells 080 transduced by the genetically-engineered viruses 020of FIG. 1A overexpress the therapeutic protein/growth factor 090 encodedby the transgenic therapeutic nucleic acid molecule (e.g., in thepresent embodiment, VEGF). Meanwhile, the stent 071 can contain emptymicrospheres 100. FIG. 1C illustrates the end of the wound healingprocess. At location 110, which was previously wounded, an healed innerlining of artery can be observed. The re-endothelialization of thepreviously denuded endothelial layer 120 is now complete by the presenceof newly generated endothelial cells 120. The fibrin layer 050(comprising empty or complete PLGA microspheres) of the stent 071 cancontinue its biodegradation.

Viral Microparticles

The present disclosure provides viral microparticles for the localdelivery of a therapeutic nucleic acid molecule. In order to mediatetheir therapeutic effects, the therapeutic nucleic acid molecules needto enter (e.g. be delivered to) the cells of the treated individual andbe transcribed/translated by the cellular machinery of the cells of thetreated individual. The viral microparticles described herein areespecially well suited to favor ordered wound healing and/or limit (orin some embodiments even prevent) pathological wound healing (such asfor example hyperplasia of cells involved in the healing of the wound)and/or for the healing of large blood vessels. The viral microparticlescan be used in a wide variety of vertebrate hosts, providing that thecells of the treated host do not allow the replication of therecombinantly-engineered baculovirus. As such below, the viralmicroparticles described herein are especially useful for the treatmentof wound in vertebrate individuals and, in some embodiments, in mammals(such as humans, dogs, horses, etc.).

The viral microparticles of the present disclosure are not limited to aparticular genera, type or subtype of baculovirus. Baculoviruses are alarge family of rod-shape viruses which have a circular double-strandedDNA-based genome. Baculoviruses have very species-specific tropismsamong the invertebrates. Even though baculoviruses are capable ofentering (e.g. infecting or transducing) mammalian cells in culture theyare not known to be capable of replication in mammalian or othervertebrate animal cells. Further, the baculovirus genome is not know tointegrate in the genome of a vertebrate host cell and is ultimatelydegraded by the host cell within a couple of days, thereby limiting thepresence of the virus in the infected vertebrate host.

The baculovirus that can be included in the viral microparticlesdescribed herein is can be from the granuloviruses (GV) genera. Suchbaculovirus usually contain a single capsid per viral envelop and isusually occluded in a granulin matrix. In an alternate embodiment, thebaculovirus which can be included in viral microparticle can be from thenucleopolyhedrovirus (NPV) genera. Baculovirus of the NPV genera containeither single (SNPV) or multiple (MNPV) nucleocapsids per envelope andare usually occluded in a polyhedrin matrix. As show below, the subtypeAutographa californica multicapsid nucleopolyhedrovirus (AcMNPV) wassuccessfully used in exemplary embodiments of the viral microparticlesdescribed herein. Baculovirus of the AcMNPV subtype are extensively usedand characterized by those skilled in the art. However, the viralmicroparticle of the present disclosure can also comprises otherbaculovirus subtypes, such as, for example Bombyx morinucleopolyhedrovirus (BmNPV).

The viral microparticles comprises a recombinantly-engineeredbaculovirus embedded in a polymeric matrix. The baculovirus' genomeserves as a vector for delivering the therapeutic nucleic acid moleculesto the cells of the treated individuals. In the gene-delivery systemdescribed herein, the baculovirus is used because of its intrinsicability to enter into the cells (e.g. infect/transduce the cells) of thevicinity of the wound (even though the cells may not be under activedivision, e.g. resting/non-dividing cells) and have the therapeuticnucleic acid molecule transduced by the cellular machinery of theinfected cells. The baculovirus is labeled “genetically-engineered”because it has been manipulated to include, within its viral genome, anheterologous therapeutic nucleic acid molecule. More specifically, thetherapeutic nucleic acid molecule has been integrated (and in anembodiment, specifically integrated), into the baculovirus genome. In anembodiment, the therapeutic nucleic acid molecule can be specificallyintegrated in the baculovirus' genome, for example, downstream of thepolyhedrin gene. As used in the context of the present disclosure, thetherapeutic nucleic acid molecule is considered “heterologous” to thebaculovirus because it is not natively found in the viral genome. Asalso used in the context of this disclosure, the term “therapeutic”,when used in conjunction with the expression “nucleic acid molecule”refers to the ability of the nucleic acid molecule to providetherapeutic benefits to the infected host and not to the baculovirusitself. In an embodiment, the transgenic/heterologous nucleic acidmolecule is derived from the species of the vertebrate host intended toreceive the transgenic/heterologous nucleic acid molecule.

In some embodiments, the therapeutic nucleic acid molecule is integratedinto the baculovirus' genome while maintaining an operative linkage (andin some embodiments a direct operative linkage) with a promoter which isgoing to be recognized and used by the cellular machinery of theintended infected/transduced host cell to allow (or increase) theexpression of the therapeutic nucleic acid molecule. The presentdisclosure is not limited to any particular promoter from any particularorigin as long as the promoter is recognized and used by the cellularmachinery of the infected/transduced host cells. Promoters endogenous aswell as heterologous to the baculovirus can be successfully used.Constitutive as well as regulated promoters can be used, depending onthe intended therapeutic use. However, as shown below, a constitutiveviral promoter (for example a viral promoter from cytomegalovirus) hasbeen successfully used to allow/increase the expression of thetherapeutic nucleic acid molecule in the infected/transduced vertebratecell.

The heterologous therapeutic nucleic acid molecule integrated into thebaculovirus genome can encode, for example, a mammalian protein, such asVEGF. The nucleic acid molecule is considered heterologous to thebaculovirus because it encodes a protein which is not natively found orexpressed by the virus. The nucleic acid molecule is also consideredtherapeutic to the infected/transduced host because it provides atherapeutic benefit, such as, for example, rapid endothelialization of astent and thereby reduces the risk of ISR in the treated host (whencompared to an untreated host).

This gene-delivery system is not limited to any particular nucleic acidmolecule (or encoded protein). As show herein, the therapeutic nucleicacid molecule can be used to increase the expression of a protein (or aplurality of proteins) to provide a therapeutic benefit and, in someembodiments, favor wound healing. In an embodiment, the heterologoustherapeutic nucleic acid molecule encodes a protein, such as a growthfactor, which will be useful for promoting wound healing. In someembodiments, especially when a blood vessel is injured or ruptured inthe wound, the growth factor can be an angiogenic growth factor. Agrowth factor is considered angiogenic when it has to ability to promotethe formation and establishment of a blood vessel. For example,angiogenic growth factors can promote endothelial cell proliferation,migration, extracellular matrix remodeling, endothelial cell tubeformation and stability (e.g. association with smooth muscle cellsand/or pericytes). Such growth factors include, but are not limited to,vascular endothelial growth factor (VEGF, including VEGF-A, PGF, VEGF-B,VEGF-C and VEGF-D), fibroblast growth factor (FGF, including FGF-1 andFGF-2) as well as angiopoietin (Ang including Ang1, Ang2, Ang3 andAng4).

However, in another embodiment, the therapeutic nucleic acid moleculedoes not need to encode a protein and can be used as a source nucleicacid transcripts (miRNA, sRNA, triplex oligonucleotides, ribozymes,etc.) which will modulate (e.g. increase or decrease) the transcriptionof specific genes in infected cells to mediate their therapeutic effectsand ultimately promote wound healing.

Since the baculovirus is used to mediate the transfer of the therapeuticnucleic acid molecule to cells in the vicinity of a wound, the virusmust be able to enter or infect the cells in the vicinity of the wound.Such cells include, but are not limited to, mesenchymal cells (such asfibroblasts, adipocytes, pre-adipocytes), stem cells (mesenchymal orother), vascular tissue cells such as endothelial and muscle cells (suchas smooth muscles cells or pericytes).

Optionally, the surface of the baculovirus that is included in the viralmicroparticle can be functionalized to increase cell penetration,increase nuclear localization and/or provide location specificity. Suchsurface functionalization can be made by covalently attaching to thesurface of the baculovirus peptides which are known to increase cellpenetration, increase nuclear localization and/or provide cellspecificity. Alternatively, the baculovirus can be geneticallyengineered to express, on its surface, peptides which are known toincrease cell penetration, increase nuclear localization and/or providecell specificity. Such peptides include, but are not limited to,cell-penetrating peptides such as TAT peptide (a transcriptionalactivator protein encoded by human immunodeficiency virus type 1(HIV-1)), bioactive ligands such as the RGD peptide as well as syntheticpolymers such as polyethylenimine (PEI) and poly-L-lysine (PLL).Alternatively, the peptide can also be used to localize the baculovirusto a specific location, for example the extracellular matrix.

Optionally, the baculovirus that is included in the viral microparticleis coated with a cationic polymer prior to its incorporation in theviral microparticle. As it is know in the art, baculoviruses have arelatively negatively charged surface which may interfere (e.g. limit)gene delivery/transduction efficiency. As such, but providing a cationic“coat” to the baculovirus surface, it may be possible, in someembodiments, to increase gene delivery/transduction efficiency. Thecationic polymer that can be used to coat the baculovirus does not needto be covalently associated to the surface of the baculovirus, it can benon-covalently associated to the surface of the baculovirus byelectrostatic/ionic interactions. The cationic polymer can be used tosubstantially completely coat the surface of the baculovirus or at leastcoat the majority of the surface of the baculovirus' envelope. In someembodiments, the cationic polymeric coat can be used to enhance theprotection the baculovirus against degradation that may occur once it isintroduced into a vertebrate host (for example, from serum).

The cationic polymer can be a linear, a branched and/or a circularpolymer. In some instances, it is beneficial to use a single type ofcationic polymer to form the coat. However, in other embodiments, it maybe necessary to use more than one type of polymers. In additionalembodiments, it is preferred that the polymer possesses at least one(and even more preferably a plurality) available amino group(s) toprovide cationic character. Alternatively, other groups other than aminocan be present to provide cationic character. The cationic polymer isalso preferably considered soluble in water. In yet another embodiment,once coated with the cationic polymer, the viral particle has a netpositive zeta potential when measured at a physiological pH (e.g.between 7.0 and 7.6, usually between 7.3 and 7.4).

Even though the cationic polymeric coat is not limited to any particularcationic polymer, as shown below, a cationic dendrimer can be used toform the baculovirus coat. As known in the art, dendrimers arerepetitively branched molecules. A dendrimer is typically symmetricaround the core, and often adopts a spherical three-dimensionalmorphology. In the Examples provided herein, a G-0 poly(amidoamine)dendrimer (or PAMAM) has been successfully used to coat the baculovirus'surface (e.g. envelop). The core of PAMAM is a diamine (commonlyethylenediamine), which is reacted with methyl acrylate, and thenanother ethylenediamine to make the generation-0 (G-0) PAMAM. Successivereactions create higher generations, which tend to have differentproperties. Other dendrimers can be used in combination of PAMAM or toreplace PAMAM.

Additional cationic polymers include polypeptide-based polymers, such ashomo-polypeptides. One homo-polypeptide of interest is poly-L-lysine (orε-Polylysine). Poly-L-lysine is typically produced as a homo-polypeptideof approximately 25-30 L-lysine residues. In contrast to theconventional peptide bond that is linked by the alpha-carbon group, thelysine amino acids of poly-L-lysine are molecularly linked by theepsilon amino group and the carboxyl group.

In some embodiment, polybrene can be used as a cationic polymer.Polybrene (1,5-dimethyl-1,5-diazaundecamethylene polymethobromide,hexadimethrine bromide) is known to be a cationic polymer used toincrease the efficiency of viral infection of certain cells. It isbelieved that polybrene acts by neutralizing the charge repulsionbetween virions and sialic acid on the cell surface.

Another cationic polymer that can be used to coat the baculovirus ispolyethyleneimine (or PEI). PEI is a polymer with repeating unitcomposed of the amine group and two carbon aliphatic CH₂CH₂ spacer. PEIcan be provided in a linear form (containing all secondary amines), inbranched form (containing primary, secondary and tertiary amino groups)as well as in dendrimeric form.

The “naked” (e.g. not coated with the cationic polymer) or “coated”(e.g. coated with the cationic polymer) baculoviruses are not introducedinto a host directly in the wound or at the vicinity of a wound. In thecontext of the present disclosure, they are embedded in a polymericmatrix to form a viral microparticle prior of being introduced into thehost. The polymeric matrix is used to release, in a controlled fashion,the baculovirus in the wound or in the vicinity of the wound.Baculoviruses formulated in such polymeric devices are released eitherby diffusion through the polymer barrier, or by erosion of the polymermaterial, or by a combination of both diffusion and erosion mechanisms.As used in the context of the present disclosure, the expression“controlled release” refers to the discharge of baculovirus in responseto stimuli and/or time. The polymeric matrix thus prolongs therapeuticbenefits but also attempts to maintain the delivery of therapeuticnucleic acid molecule within the therapeutic window to avoid potentiallyhazardous peaks in baculovirus concentration/transgene expressionfollowing administration.

In addition, although the term viral “microparticles” is used throughoutthe present text, it is not limited to a particular shape or size ofparticles. It encompasses particles which are considered microspheres,microcapsules, nanocapsules as well as nanospheres. The relative size ofthe viral microparticles can be in the micromolar range. In anembodiment, the relative size of the microparticle is at least 5 μm, 6μm, 7 μm, 8 μm or 9 μm and/or at most 10 μm, 9 μm, 8 μm, 7 μm or 6 μm aswell as any combinations therefrom (for example from 5 μm to 10 μm). Inthe embodiment in which the viral microparticles are of a relativelyspherical shape, the relative diameter of the microparticle is at least5 μm, 6 μm, 7 μm, 8 μm or 9 μm and/or at most 10 μm, 9 μm, 8 μm, 7 μm or6 μm as well as any combinations therefrom (for example from 5 μm to 10μm).

Consequently, because the polymeric matrix of the microparticle needs tobe degraded to allow the discharge of the genetically-engineeredbaculovirus, it has to be biodegradable (e.g. have the intrinsic abilityto be cleaved/destroyed within the host). In an embodiment, thepolymeric matrix is capable of being degraded/eroded by water. Further,because the polymeric matrix needs to be introduced into a host, it alsohas to be biocompatible (e.g. have the ability to perform its desiredfunction without eliciting any undesirable local or systemic effects inthe host, such as, for example, immune rejection or inflammation). Sincethe polymeric matrix is going to be used to embed thegenetically-modified baculovirus, it should preferably be made from apolymeric material which has the ability to form a matrix underconditions which will not interfere with the infectivity/integrity ofthe baculovirus. Depending on the required biodegradability of the viralmicroparticle, the matrix of the viral microparticle can be made from asingle type of polymer or more than one of polymers. Further, in someembodiment, and depending on the type of polymeric material used, thepolymer of the matrix can be cross-linked to increase thestability/rigidity of the viral microparticle.

Even though the matrix of the viral microparticle is not limited to anyparticular type of polymer, as shown below in the Examples section,polyesters can be success used to produce such matrix. The polyestersthat can be used can be, for example, of synthetic nature and have, inyet another embodiment, an aliphatic main chain. In the Examples below,poly(lactic-co-glycolic acid) (also referred as PLGA) has been used toform the matrix of the viral microparticle. PLGA is a polyester whichcan be conveniently degraded through the hydrolysis of its esterlinkages in the presence of water.

However, in the context of the present disclosure, it is alsocontemplated that other biodegradable polyesters can be used, alone orin combination with PLGA). Such alternatives polyesters include, but arenot limited to polycaprolactone (or PCL), poly(glycerol sebacate) (PGS)and polylactide (or PLL).

As indicated herein, the baculoviruses are embedded within the polymericmatrix. The baculovirus are embedded at least partially (and in someembodiments entirely) within the matrix. It is assumed that the type ofbaculovirus embedment will vary depending on the nature of polymericmatrix as well as on the process for making the viral microparticles. Insome embodiments, the baculovirus is completely embedded in thepolymeric matrix such that the external elements of the baculovirus'envelop are in direct contact with the polymeric matrix. Incomplementary or alternative embodiments, the baculovirus is partiallyembedded in the polymeric matrix such that a portion of the externalsurface of the baculovirus' envelop is located inside the polymericmatrix and another portion of the external surface of the baculovirus'envelop is protruding away the polymeric matrix. It is contemplated thatsome viral microparticles will comprises only completely embeddedbaculovirus, other viral microparticles will comprise only partiallyembedded baculovirus whereas other viral microparticles will compriseboth completely and partially embedded baculovriuses.

As indicated above, the size and shape of the microparticle will dependon the properties of the polymeric material to make the matrix as wellas the process used for making the viral microparticles. In someembodiments, the viral microparticles will have a relatively sphericalshape (even though embedded baculovirus may protrude from the surface ofthe microparticle). In other embodiments, the viral microparticles havea relative diameter of at least 5 μm. In another embodiment, the viralmicroparticles have a relative diameter of around 10 μm, depending onthe solvents used for the preparation such as water/oil/water emulsionmethod or water/oil/oil emulsion, as well as based on the method such ashomogenization or sonication.

The viral microparticles can be directly introduced into a wound or inthe vicinity of a wound to mediate their therapeutic effect. The viralmicroparticles can be physically located to a wound or its vicinity viadeposition or injection for example.

Process for Making Viral Microparticles

Depending on the type of polymeric material used to form the viralmicroparticle's matrix, various processes can be used to make the viralmicroparticle. However, as shown below, it is beneficial, in someembodiment, to make the viral microparticles as a water-in-oil-in-oilemulsion to preserve the integrity/limit the damage of the embeddedbaculovirus.

An embodiment for making the viral microparticles is shown in FIG. 1D.In a first (optional) step, the genetically-engineered baculoviruses 140can be admixed with a cationic polymer 150 (such as, for example,G0-PAMAM dendrimer) to form a complex 160. The complex 160 is made up ofgenetically-engineered baculoviruses 140 and the cationic polymer 150exhibiting ionic interactions with one another. The surface charge ofthe complex 160 is less negative than the surface charge of the “naked”baculovirus 140. In a second step, the genetically-engineeredbaculoviruses 140 (not shown) or the complexes 160 (shown on thisfigure) are introduced into an aqueous solution to form an aqueous phase170. In some embodiments, this aqueous solution can be an aqueous saline(for example a phosphate-buffered saline) optionally comprising isotonicand/stabilizing agents such as glycerol and/bovine serum albumin. Afirst oil phase 180 is also provided by combining polymerizablebiodegradable and biocompatible polymers (e.g., PLGA in this embodiment)in a water immiscible and volatile organic solvent (e.g.,dichloromethane in this embodiment) which can optionally contain asurfactant (e.g. acetonitrile in this embodiment). A water-in-oil (w/o)emulsion is prepared by combining the water phase 170 and the oil phase180 in an homogenizer 190. The water phase 170 and the oil phase 180 arehomogenized until a water-in-oil emulsion 200 is formed. In thewater-in-oil emulsion 200, the genetically-engineered baculoviruses arelocated in the water phase of the emulsion. A water-in-oil-in-oilemulsion is then prepared by combining the water-in-oil emulsion 200with a second oil phase (not shown, containing a biocomptaible oil (forexample a vegetable oil e.g., corn oil) and a surfactant (Span 80)) andstirring or homogenizing the resulting mixture 210 with an homogenizer190. The water-in-oil emulsion 200 and the second oil phase aresubmitted to homogenization until a water-in-oil-in-oil 220 emulsion isformed. The baculoviruses are located in the water phase of thewater-in-oil-in-oil 220 emulsion. The water-in-oil-in-oil emulsion 220can be then left to stand allow the hardening of the viral microspheresand the evaporation of the water immiscible and volatile organic solventof the resulting mixture 230. Optionally, the viral microspheres 240 canbe centrifuged washed and collected.

One of the processes for making the viral microparticles describedherein first comprises resuspending, in an aqueous solution, thegenetically-engineered baculovirus. The aqueous solution can compriseelements which help maintaining the integrity of the viral particles,such as for example an isotonic and buffered solution (e.g. a phosphatebuffered saline containing glycerol and/or a stabilizing agent (forexample bovine serum albumin)). As indicated above, the baculovirus canbe “naked”, “coated” with a cationic polymer and/or“surface-functionalized” prior to its resuspension in the aqueoussolution.

The resulting aqueous preparation (e.g. resuspended baculovirus) is thencombined with a solution of the biodegradable biocompatible polymer(which will eventually form the matrix of the viral microparticle) andmixed to obtain a water-in-oil (w/o) emulsion. The biodegradablebiocompatible polymer can be polymerizable to form a polymeric matrix.To favor the emulsion, in an embodiment, a surfactant (such as forexample acetonitrile, poly(vinyl alcohol) (PVA) and/or poly(vinylpyrrolidone) (PVP)) can be used. The surfactant is preferably added tothe oil phase prior to the emulsion. Due to the chemical nature of thebiodegradable biocompatible polymer, it is necessary to solubilize it ina water immiscible, volatile organic solvent to provide a single phaseprior to combining it to the resuspended baculovirus aqueouspreparation. For example, when PLGA is used as the biodegradablebiocompatible polymer, a wide variety of water immiscible, volatileorganic solvents can be used. Such solvents include, but are not limitedto chlorinated solvents (such as, for example dichloromethane (DMC)),tetrahydofuran, acetone or ethyl acetate In addition, even thoughvarious techniques are known to those skilled in the art to achieve thew/o emulsion, in order to preserve the integrity/infectivity of thebaculovirus, it may be beneficial to use an homogenization methodinstead of more disruptive ones (sonication for example).

Once the water-in-oil emulsion is formed, it is combined and mixed withan oil to form a water-in-oil-in-oil (w/o/o) emulsion. For this secondemulsion, any type of biocompatible oil can be used (e.g. vegetable oilsuch as corn oil for example). In some embodiment, the oil can besupplemented with a biocompatible non-ionic surfactant (e.g. Span 80™for example) prior to the second emulsion. Even though varioustechniques are known to those skilled in the art to achieve the w/o/oemulsion, in order to preserve the integrity/infectivity of thebaculovirus, it may be beneficial to use an homogenization techniqueinstead of more disruptive ones (sonication for example).

Once the w/o/o is achieved, the solution can be stirred under conditionsto allow the evaporation of the water immiscible, volatile organicsolvent and, ultimately, the formation of the hardened viralmicroparticles. In embodiments, it may be necessary to recuperate theviral microparticles and wash them to remove additional impurities (suchas, for example, unembedded baculovirus).

The process described herein can be completed at ambient temperature.

Pharmaceutical Compositions of Viral Microparticles

Even though the viral microparticles can be used on their own and placeddirectly within a wound or in the close vicinity of a wound, in someembodiment, it can be beneficial to provide the viral microparticles inthe form of a pharmaceutical composition. Besides comprising the viralmicroparticles described herein (or produced by the process describedherein), the pharmaceutical composition also comprises a componentcapable of forming a matrix in which the viral microparticles aredispersed. Depending on the intended use, the viral microparticles canbe relatively homogeneously or heterogeneously dispersed within thematrix of the pharmaceutical composition. The matrix can also serve tocontrol the release of the viral microparticles at the vicinity of awound.

In one embodiment, the pharmaceutical comprises a gelling agent as acomponent capable of forming a matrix. The gelling agent is capable offorming a gel (e.g. a cross-linked system which exhibits no flow when inthe steady-state) in which the viral microparticles are dispersed. Inthe pharmaceutical composition, the gelling agent can be in a liquid ora solid form. However, once introduced into the vicinity of a wound (forexample in a cavity formed by the wound), the gelling agent ispreferably in a solid form to limit displacement of the pharmaceuticalcomposition away from the wound, and ultimately allow the local deliveryof the baculovirus. The gelling agent is intended to be biocompatiblewith the treated vertebrate individual as well as being capable ofpreserving the integrity of the viral microparticles.

In some advantageous embodiments, the gelling agent is capable offorming a hydrogel (a gel comprising water molecule(s)). In additionalembodiments, the gelling agent can comprise a cross-linkable protein,and in even a further embodiment, a cross-linkable protein derived froma clot (fibrinogen for example) and/or the extracellular matrix(collagen, laminin, elastin, fibronectin for example). In alternativeembodiments, the gelling agent can be a non-proteinaceous element (orcombination thereof) of the extracellular matrix (glucoasminoglycan(heparan sulfate, chondroitin sulfate, keratan sulfate), hyaluronic acidfor example). In further embodiments, the gelling agent can be asynthetic nanomaterial such as, for example, a polymeric nanoscaffoldand/or a nanoporous material. The gelling agent can also comprise morethan one type of gellable entity. The type of gelling agent that can beincluded in the pharmaceutical composition is not limited to theexemplary embodiments described herein. For example, the gelling agentmay also include be collagen, gelatin, extracellular matrix basedhydrogels, covalently or ionically conjugated hydrogels, composite orhybrid hydrogel structures, photocrosslinkbale hydrogels as well ascombinations thereof.

As indicated above, the pharmaceutical composition can be provided in aliquid form. In such embodiment, it will be necessary to convert thepharmaceutical composition into a solid form either prior toadministering it to the vertebrate individual or upon administration tothe vertebrate individuals. In embodiments in which the gelling agentneeds to be cross-linked with a cross-linking agent (for examplefibrinogen and its associated cross-linking agent thrombin), thepharmaceutical composition can be provided as a kit comprising thegelling agent (optionally already comprising the viral microparticles)and the cross-linking agent.

In embodiments in which the gelling agent needs to be heated to allowthe formation of a solid composition, the pharmaceutical composition(optionally already comprising the viral microparticles) can be providedwith instructions on the conditions to be used to achieve a solid form.

As also indicated above, the pharmaceutical composition can also beprovided in a solid form which is ready to be administered to thevertebrate individual in need thereof. In such embodiment, thepharmaceutical composition preferably already comprises the viralmicroparticles dispersed therein.

In order to formulate the pharmaceutical compositions described herein,it is possible to combine the gelling agent and the viralmicroparticles. In an embodiment, the gelling agent is in a liquid formwhen it is first admixed with the viral microparticles and thentransformed into a gel (which can optionally include a step of adding across-linking agent to the mixture and/or submitting the mixture to aheat treatment).

Viral Microparticles Comprising Supports

In order to administer the viral microparticles described herein, it ispossible to add/include them to a biocompatible support and introducesuch support in the wound or its vicinity.

Alternatively, it is also possible to apply the pharmaceuticalcomposition onto or within the support. When the pharmaceutical isprovided in a solid form, it is possible to attach it on the support bymechanical means (a suture or a clip for example) or add a linker toincrease the adhesiveness of the pharmaceutical composition to the solidsupport (e.g. fibrin glue for example). When the pharmaceuticalcomposition is provided in a liquid form, it is possible to apply it ona support (e.g. coating, dipping, rolling, brushing, etc.) and cause thepharmaceutical composition to gel on the surface of the support. Oneexemplary embodiment of such process included the coating of the supportwith a fibrinogen mixture comprising the viral microparticles and thesubsequent coating of the support with a thrombin solution which causesthe formation of a fibrin gel (comprising the viral microparticles) onthe support.

In the context of the present disclosure, it is not necessary to coatentirely the support to observe a beneficial effect for the treatedvertebrate individual. In some embodiments, it may be sufficient andeven advantageous to coat only a portion of the surface with the viralparticles (formulated or not in the pharmaceutical composition) of thesupport prior to its introduction in the treated vertebrate individual.For example, when a stent is to be coated with the viral particles, itmay be advantageous to coat the surface of the stent in contact with theintima (exterior surface) and limit the coating of the surface of thestent in contact with blood circulation (interior surface) to avoidsystemic dissemination of the viral microparticles. Supports which canbe modified to include the viral microparticles described herein(formulated or not in the pharmaceutical composition) are preferablysolid supports which are designed to be located in a wound or in thevicinity of a wound. These supports includes, but are not limited to,dressings, fasteners, sealants, insertion tools, catheters, stents,tubes, adhesives, etc. The supports can be made of biocompatiblepolymers, nanomaterials and/or metal.

Once included on a support, the viral microparticles are relativelystable and the support can be stored for a couple of months withoutobserving a substantial decrease in viral infectivity. As shown in theExamples section below, a stent coated by a fibrin gel comprising viralmicroparticles was stored for three months at 4° C., −20° C. or −80° C.without observing a substantial decrease in viral infectivity. If asupport comprising viral microparticles is intended to be stored priorto its introduction in a vertebrate host, it may be preferred to storeit at −80° C. to limit a decrease in viral infectivity.

Therapeutic Applications of Viral Microparticles

As indicated above, the viral microparticles described herein arecapable of mediating therapeutic effects to favor thenon-pathological/homeostatic wound healing process and/or limitspathological wound healing in a treated invertebrate individual. Theviral microparticles mediate their therapeutic actions only in thedirect environment in which they localize and only for a limited timewindow. As discussed herein, the baculovirus of the viral microparticlescannot replicate in the vertebrate host and lack the intrinsic abilityto disseminate in remote areas (if they do not enter the generalcirculation). As such, the baculovirus can only mediate its therapeuticactions locally (in opposition to systemically or remotely) to the areain which they are introduced. As also discussed herein, the baculovirus'genome is eventually degraded in the infected host and as such can onlymediate its therapeutic actions during a specific time window whichallows a transient expression of the therapeutic nucleic acid molecule.

As used in the context of the present disclosure, the term “wound”refers to a site of injury or damage which causes the disruption ofnormal continuity of structures (such as, for example, the epitheliumand/or the endothelium). In some embodiments, a wound is restricted toinjuries/damages caused by physical means (the introduction of a stentin a large vessel for example). In addition, an “homeostatic woundhealing” refers to a process restoration of integrity to injured tissueswith cellular and extracellular matrix remodeling. Further, “apathological wound healing” condition refers a condition in which thedisorderly resolution of a scar has a pathological effect, for example,hypertrophic scarring, restenosis, neointima hyperplasia formation, etc.

The viral microparticles described herein can be, in embodiments, use topromote wound healing, prevent a condition associated with apathological wound healing as well as alleviation of symptom(s)associated with wound healing. These expressions(promote/prevent/alleviate) refer to the ability of the method describedherein or the viral particles to limit the pathological development,progression and/or symptomology of wound healing. Broadly, thetherapeutic methods can encompass, in embodiments, the increase in theproliferation of some cells (for example endothelial cells), thereduction of proliferation of other cells (e.g. for example mesenchymalcells (such as fibroblasts) or muscle cells (such as smooth muscle cellsor pericytes), the modulation of extracellular matrix cleavage andproduction and/or the modulation of inflammatory processes associatedwith the wound.

In order to achieve a therapeutic benefit to the treated individual, itis preferable to administer a pharmaceutically effective amount ortherapeutically effective amount of the viral microparticles to thetreated individual. These expressions refer to an amount (dose)effective in mediating a therapeutic benefit to a vertebrate individualIt is also to be understood herein that a “pharmaceutically effectiveamount” may be interpreted as an amount giving a desired therapeuticeffect, either taken in one dose or in any dosage or route, taken aloneor in combination with other therapeutic agents.

The present disclosure thus provides a method of favoring the healing ofa wound in a vertebrate individual in need thereof. In such therapeuticmethods, the viral microparticles are first inserted within a wound (orin the vicinity of a wound) in a vertebrate individual. The viralmicroparticles can optionally be formulated in the pharmaceuticalcomposition described herein and/or be included in the solid support.The viral microparticles are preferably located in the vicinity of thewound such that the recombinantly-engineered baculovirus can bedischarged in the vicinity of their target cells and mediate thetransduction of the therapeutic nucleic acid molecule in the infectedhost cells.

The method can be applied in any injury tissue (skin, large vessel, eye,etc.) where orderly wound healing is warranted. The methods can also beapplied to any vertebrate individuals (such as mammals, and in someembodiments, such as humans), provided that their cells do not allow thereplication of the genetically-engineered baculovirus.

In some embodiments, and as shown in the Examples below, it can bebeneficial to use, as a therapeutic nucleic acid molecule, a moleculeencoding a growth factor and may have angiogenic properties favoringangiogenesis in the vicinity of the wound. In the context of the presenttherapeutic methods, the term “angiogenesis” is associated with therestoration of appropriate blood supply to the wounded tissue and doesalso encompass favoring the endothelialization of the support that isbeing introduced into the treated individual. Several diseases, such asischemic chronic wounds and in-stent restenosis, are the result offailure or insufficient blood vessel formation and may be treated by alocal expansion/maturation of blood vessels. In some embodiment, theviral microparticles-containing support, by preventing ISR can alsoreduce the need (and in some embodiments eliminate the need) foranti-platelet therapy in treated individuals. In some additionalembodiments, the viral microparticles-containing support can be used forcarotid artery stenting. Further, therapeutic angiogenesis may bewarranted to treat a variety of atherosclerotic diseases, like coronaryheart disease, peripheral arterial disease, wound healing disorders,etc.

In some further embodiments, the viral microparticles described hereincan be formulated as a topical composition (such as for example atopical cream, shampoo or conditioner). In such embodiment, the viralmicroparticles can be used to stimulate skin wound healing, skinregeneration and/or hair growth for example.

In yet another embodiment, the viral microparticles described herein canbe used in tissue engineering applications. In such embodiment, theviral microparticles can be formulated with extracellular matrixcomponents or a combination of different extracellular matrix components(which may or may not be biodegradable) and used in various tissueengineering applications, such as, for example, reconstructive soft andhard tissues (e.g., skin and bone).

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the disclosurerather than to limit its scope.

Example I Material and Methods

The mammalian expression vector pCI and pVL1392 transfer vector wereprocured from Promega and BD Biosciences respectively. pCMV-XL4mammalian expression vector carrying human hVEGF cDNA was obtained fromOrigene Technologies (Rockville, Md.). The pVL1392 vector and pCIvector, which harbors the pCMV promoter, were digested with BglII andBamH1 restriction enzymes. The cut out pCMV gene and digested pVL1392vector were purified and a basic ligation reaction with T4 DNA ligase(Promega) was performed to insert the pCMV gene into the pVL1392transfer vector by directional cloning method. The pVL1392-PCMVconstruct was then linearized by Noti enzyme digestion. It was thenligated in the similar way to the hVEGF cut out of the Noti digestedpCMV-XL4-hVEGF plasmid to form pVL1392-pCMV-hVEGF as the final transfervector construct. The recombinant transfer vector was then transformedinto the high efficiency DH5α-competent E. coli (Invitrogen) by heatshock method for amplification. The plasmids were then purified withQiaprep spin Miniprep™ kit (Qiagen Sciences, MD). The recombinant hVEGFbaculoviruses (BacVEGF) were generated by cotransfection of Sf9 insectcells with linearized baculovirus DNA (BD Baculogold) along withpurified recombinant transfer vectors using Cellfectin™ (Invitrogen LifeTechnologies, Carlsbad, Calif.) transfection reagent as mentioned inearlier study (Paul A et al. 2011). The recombinant baculovirus stock(BacVEGF) was harvested 72 hours post transfection and further amplifiedusing routine procedures. The viral titer [plaque forming unit (pfu)/mL]of the amplified viral stock was then determined using the BaculovirusFast Plax Titer™ Kit (Novagen, Madison, Wis.) according to themanufacturer's protocol. Similarly, baculoviruses carrying MGFP gene(BacMGFP) and no transgene (BacNull) were generated as mentioned inprevious work (Paul A et al. 2011).

Preparation Methods for Virus Encapsulation:

w/o/o and w/o/w. In order to encapsulate the viral particles bywater-oil-water (w/o/w) double emulsion and solvent evaporation method(Matthews C B et al. 1999, Mok H et al. 1999), 5×10¹³ pfu wasresuspended in 100 μl of PBS containing 10% glycerol and 50 mg/ml BSA.This primary w/o emulsion solution was prepared by homogenizing theabove mixture in 1 ml of dichloromethane (DCM) containing 50 or 100 mgof PLGA (poly(D,L-lactic-co-glycolic acid) for 1 min at 10 000 rpm usingPowerGen™ Homogenizer 125 (Fisher Scientific). The resulting primaryemulsion was added to 3 ml of 10% polyvinyl alcohol (PVA) andhomogenized for 3 min at 14 000 rpm to form the secondary w/o/wemulsion. This solution was further agitated with a magnetic stirringbar in 10 ml of 1% PVA for 4 h to evaporate the dichloromethane. Thehardened PLGA microspheres were centrifuged at 9000 g for 10 min, washedthrice with PBS and resuspended in fibrinogen solution for furtherexperiments.

In order to encapsulate the viral particles by w/o/o double emulsion andsolvent evaporation method (Lee J H et al. 2000), 5×10¹³ pfu wasprepared as mentioned above. This primary w/o emulsion solution washomogenised in 1 ml of DCM containing 50 or 100 mg of PLGA and 1 mlacetonitrile for 1 min at 10 000 rpm. The resulting primary emulsion wasadded to 5 ml of corn oil containing 2% Span 80™ and homogenized for 3min at 14 000 rpm to form the secondary w/o/o emulsion. This solutionwas further agitated with a magnetic stirring bar in 10 ml of corn oilcontaining 2% Span 80™ for 4 h to evaporate the DCM. The hardened PLGAmicrospheres were centrifuged at 9000 g for 10 min, washed thrice withPBS and stored temporarily at 4° C. The entire mechanism of PLGAmicroencapsulation of virus particles are demonstrated schematically inFIG. 1B. To visualize and surface-characterize, the microspheres weremicrophotographed with scanning electron microscope (SEM; Hitachi S-4700FE), as well as with Atomic Force Microscope (AFM) with a Nanoscope III(Digital Instruments, USA) using a silicon cantilever in tapping modeand nanoscope v 5.12r5 image analysis software. To view the innerstructure of the virus-loaded microspheres, the dried PLGA microsphereswere exposed to OsO₄ vapor at room temperature for 24 h in the presenceof 1% OsO₄ solution, and then dipped in an epoxy matrix, cured at roomtemperature for 24 h and microtomed. The ultrathin cross-sections werenoticed using Transmission Electron Microscope (TEM; Philips CM200FEG-TEM).

The encapsulation efficiency of Bac within PLGA microspheres wasdetermined by digesting the PLGA polymer with 1N NaOH for 24 h at 4° C.to extract the Bac particles into the aqueous solution (Mok H et al.2007). The titer of Bac was then determined by a standard Bac titerassay. As an experimental control, another set of experiment was carriedout where the free Bac were treated in the similar way with 1N NaOHbefore determining the viral titer. The encapsulation efficiency wasthen calculated by dividing the titer of encapsulated viral particles bythe initial titer of viral particle used. All experiments were carriedout in triplicates.

Formulation of Bac-PAMAM Microparticles.

PAMAM dendrimer (generation 0) with ethylenediamine core (MW: 516.68)containing 4 surface primary amino groups was procured from SigmaChemicals and resuspended in phosphate buffered saline (PBS). Theconcentration of PAMAM peptide stock solution was initially adjusted to10 μmol in PBS solution. In order to form the PAMAM-baculovirusmicroparticle, the solutions of the PAMAM and baculoviruses were firstbrought to room temperature and adjusted to desired concentrations. Thenthe PAMAM solution and baculovirus solution were mixed according to adesired PAMAM/virus ratio (0, 0.01 μmol, 0.1 μmol, 0.5 μmol and 1.0 μmolPAMAM molecules per 10⁸ baculovirus). The mixture was incubated at roomtemperature for 30 min to form complexes, with gentle vortexing fromtime to time. The mixture was further centrifuged at 24 000 rpm for 45min, and the pellet containing the heavier Bac-PAMAM microparticle wascollected, leaving the unreacted excess dendrimer in the supernatant.The collected Bac-PAMAM was washed twice using PBS using the samecentrifugation process. For every experiment, the preparation wasfreshly made.

Stent Coating Formulation.

Firstly, the baculovirus was coated with PAMAM (0.5 μmol) and formulatedin PLGA microspheres by w/o/o method. The prepared PLGA microsphereswere resuspended in 5 mg/ml of bovine plasma derived fibrinogen,supplemented with aprotinin (20 μg/ml) to reduce fibrin degradation, andloaded in a 3 ml syringe with a 0.2-mm nozzle. The balloon expandablebare metal stainless steel stents with basic dimensions of 16 mm×3.5 mm(Liberte Monorail stent, Boston Scientific, Mississauga, Ontario) wasfirst mounted on a PTFE (polytetrafluoroethylene) mandrel that wasdriven by a rotator. The loaded aqueous fibrinogen mixture in thesyringe was then gradually drip-coated on the surface of the mountedstent layer by layer (0.2 ml of Bac loaded aqueous fibrinogen perlayer). In between every layer, 0.05 ml of thrombin solution (20 U/ml)was added all over the stent surface using a micropipette with 200 μlmicropipette tip and waited for 15 min to form thin fibrin gel layer.Polymerization of the fibrin occurred around the stent which completelyencased the stent (FIG. 1). The stent was subsequently coated with a toplayer of fibrinogen (2.5 mg/ml) cross-linked with genipin (0.045 mg/mLfinal) followed by polymerization with thrombin. The process resulted ina microsphere impregnated fibrin coated stent loaded with 5×10¹² pfuBac. The device was then crimped onto standard collapsed angioplastyballoon and delivered to the femoral artery. To visualize the PLGAmicrospheres impregnated on the fibrin coated stent surface (without thetopmost fibrin/genipin layer) under fluorescence microscope (NikonEclipse TE2000-U), 1 mg Nile red was mixed in the PLGA/dichloromethanesolution before preparing the microspheres and fabricating them on thestent. Similarly, the stents were also observed under SEM to envisagethe surface topography. As control for in vitro transduction study, freevirus containing fibrinogen complex were coated on the stents.

In Vitro Transduction Via Stent.

The stents with different formulations were crimped on the ballooncatheter and inflated in scintillation vials containing 5 ml ofphosphate buffer saline (PBS) solution (pH 7.4) with 9 atm pressure. Theexpanded stent was incubated at 37° C. for 24 h, with constant agitationat 100 rpm. The PBS solutions containing the released viruses werecollected at 12 h, 18 h and 24 h post incubation.

The 12 h incubation buffer was diluted and added at MOI of 250 to 1×10⁶seeded human aortic artery smooth muscle cells (HASMCs; Sciencell,Carlsbad, Calif., USA) after removing the standard smooth muscle cellmedia (SMCM) provided by the supplier. The 18 h and 24 h incubationbuffers were similarly diluted with same dilution factor and added toHUASMCs. After incubating the cells with the stent incubation buffer for8 h, the buffers were aspirated and replenished with complete growthmedium for 24 h. After fixation, microphotographs were taken and thetotal cell numbers were determined per 200× magnification under brightfield. GFP-expressing cells were also visually counted in the samefields, and results reported as the percentage of cells transduced(mean±SD) of at least five fields per culture in triplicate cultures.

To check the inactivation effect of serum on baculovirus present on thestent surface, the virus loaded stent with different formulations wereincubated in 50% FBS or PBS for 1 h. This was followed by addition ofthe viruses resuspended in fresh PBS to 2×10⁴ HASMCs per well in 96 wellplate with an MOI of 250 for checking the GFP expression as mentionedabove. 24 h post transduction, the fluorescein expressions per well werequantified using plate reader Victor3™ Multi Label Plate Counter (PerkinElmer, USA) in terms of normalized percentage GFP expression. Theexperiments were performed with triplicates.

To check the effect of storage conditions on stent bioactivity, thestents were stored in cryovials for 3 months at different temperatures(4° C., −20° C. and −80° C.). As control, freshly prepared stent wasused. After thawing, the stents were balloon expanded and incubated for24 h in PBS solution in scintillation vials as mentioned earlier. ThePBS solution was collected from the release vials and added to 2×10⁴HASMCs per well in 96 well plate with an MOI of 250 as mentioned above.The fluorescein expressions after 24 h were detected in plate reader andpresented as normalized percentage GFP expression.

In vitro transduction via BacVegf-PAMAM loaded stent, VEGF releasekinetics from transduced cells. In order to investigate the Vegf releasekinetics from transduced HASMCs, three types of stents (BacVegf-PAMAM,BacVegf only and ctrl stent with no virus) were expanded in PBS solutionusing balloon catheter and incubated for 24 h as illustrated earlier.The PBS solution containing the released viruses were collected andadded to 1×10⁶ HASMCs seeded per well in 6 well plate with an MOI 500.This was followed by 8 h incubation at 25° C. with subsequentreplenishment of the incubated cells with fresh culture media. Theconditioned media were collected on day 0, 2, 4, 9, 12 and 15post-transduction and quantified for Vegf expression using hVegf ELISAkit (R&D Systems).

HUVEC proliferation assay. For the cell proliferation assay, 2×10⁴ HumanUmbilical Endothelial Cells (HUVECs)/well were seeded in triplicate foreach sample in 96-well plates. After culturing for 8 h in standardendothelial cell media (ECM), the cells were washed twice with PBS. 0.1ml of CM from transduced HASMCs (Day 4 CM from BacVegf-PAMAM, BacVegfonly and ctrl stent with no virus groups) with and without hVegfantibodies (Ab) along with 0.1 ml of fresh ECM without cell growthsupplements were added to the corresponding set of wells. Similarly, CMfrom unstimulated control group mixed with fresh ECM was taken as thecontrol group. After 3 days of culture, absorbance was measured at 490nm using Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay(Promega) in a plate reader as mentioned previously (Paul A et al.2011). This assay was also used to measure the cytotoxic effects ofstent-released baculoviruses towards HASMCs.

Wound Healing Assay.

HUVECs were seeded into six well plates and grown to confluency. After24 h of serum starvation, the monolayer was carefully mechanicallywounded with a 200 μl pipette tip. The wells were then washed twice withPBS to remove the cell debris and the seeded cells were replenished with0.1 ml of the fresh ECM (without cell growth supplements) and 0.1 ml ofthe CM from different groups (CM from day4 BacVegf, BacVegf-PAMAM andctrl unstimulated group), in presence or absence of 1 μg/mL Vegfantibody. Following HUVEC migration for 12 h, the cells were fixed with4% paraformaldehyde and stained with crystal violet. The wound healingwas visualized under inverted bright field microscope andmicrophotographs with 100× magnification were taken. The number of cellswhich had moved across the starting line (mean±SD; n=3) in each groupwas assessed and analyzed using Image J software to measure the woundhealing as mentioned earlier (Paul A et al. 2011).

HUVEC Tube Formation Assay.

In vitro angiogenesis assay was performed using Cell Biolabs EndothelialTube Formation Assay as mentioned elsewhere (Jiang J et al. 2011).Briefly, 50 μl of ECM gel prepared from Engelbreth-Holm-Swarm (EHS)tumor cells were added to the 96-well plate and incubated for 1 h at 37°C. to allow the gel to solidify. 2×10⁴ cells suspended in CM fromdifferent groups (Day 4 CM from BacVegf-PAMAM, BacVegf only and ctrlstent with no virus groups), in presence or absence of Vegf antibodywere seeded per well. After 18 h incubation period, media were removedand the cells were then incubated with 50 μL of 1× Calcein AM for 30 minat 37° C. The cells were washed twice with 1×PBS and the endothelialcapillary-like tube formation in each well was examined using afluorescent microscope under 100× magnification and the HUVEC-madecapillary network was analyzed by Image J software. The results werequantified as the mean relative tubule length/view field±SD, takingtotal tubule length/view field from control group as 100.

In Vivo Surgical Procedures for Arterial Injury and Stent Implantation.

All procedures were in compliance with the Canadian Council on AnimalCare and McGill University animal use protocol, following all theethical guidelines for experimental animals. Adult beagle dogs (MarshallFarms, North Rose, N.Y.) weighing 9.5 to 11 kg were used in this study.

One week before the surgery the animals were treated daily with aspirin(325 mg/day) along with normal 21% protein dog diet (Harlan, Montreal,Canada) to avoid thrombosis. A total of 28 stents were implantedbilaterally in deep femoral arteries of 14 dogs in a randomized, blindedfashion following denudation of the arterial endothelial wall, withcontralateral arteries receiving stents from different groups. Theanimals were divided into three groups: fibrin coated stent loaded withmicroencapsulated PAMAM-BacVEGF virus [Coated (+); n=11 stents pergroup), fibrin coated stent loaded with microencapsulated PAMAM-BacNuIIvirus [Coated (−); n=11 stents per group] and bare metal stent(Uncoated; n=8 stents per group). To confirm the transgene delivery andre-endothelization, animals from first two groups were sacrificed (n=3)after 2 weeks and arteries were harvested. The remaining animals weresacrificed after 6 weeks for further analysis.

On the day of the stent implantation, after subcutaneous pre-anestheticmedication with butorphanol (0.2 mg/Kg), acepromazine (0.125 mg/Kg) andatropine (0.025 mg/Kg), the dogs were subjected to general anesthesiawith sodium pentobarbital (20 mg/Kg, injected intravenous via catheteralong with the lactated Ringer's fluid) and endotracheal intubation formechanical ventilation and isoflurane (2-3% along with supplementaloxygen) for anesthetic maintenance. Denudation of the femoral arterialendothelial layer and stent implantation was performed according to theprocedure performed in earlier studies (Welt F G et al. 2000 and WorseyF G et al. 1993). Briefly, the dog, placed in supine position underanesthetized condition, was administered intravenously with heparin (30U/Kg) as blood thinner. Under sterile conditions, the superficialfemoral arteries on both legs were surgically exposed and held inposition by surgical clips. Continuous monitoring of blood pressure(iBP), respiration, temperature, pulse oximeter oxygen saturation(SpO₂), hemodynamic and surface electrocardiograms were performedthroughout the experimental procedure using patient monitoring system(Bionet Vet, QST Technologies, Singapore). The artery lumens wereflushed with PBS to avoid mixture with arterial blood. On the basis ofangiograms, femoral segments with comparable diameters were selected onboth legs so that the stent-to-artery diameter ratio remainsapproximately 1.3. After an arteriotomy, a fogarty arterial embolectomyballoon catheter (Edwards Lifesciences Canada Inc, Ontario) was infusedthrough the saphenous arteries and advanced to the two preselectedfemoral segments and secured with a tie as mentioned elsewhere (Newman KD et al. 1995). This was followed by severe balloon injury of the innerlumen of the artery with inflated balloon (balloon/artery ratio 1.2:1)to induce endothelial abrasion. Eventually the balloon catheter mountedstents were inflated at the sites of endothelial damage in the femoralarteries with nominal pressure (9 atm) for 1 min and was then slowlywithdrawn leaving the stent in place. After stent deployment and closureof the arteriotomy site with 7-0 prolene suture, doppler ultrasoundprobe was used to check the vessel patency and confirm normal blood flowthrough of the stented arteries. The experimental animal is thenextubated and administered with buprenorphine (0.02 mg/Kg,subcutaneously) analgesic for 1-2 days. 3 animals were euthanized byoverdose of sodium pentobarbital (200 mg/Kg), intravenous at week 2,while the remaining at week 16.

Analysis of Transgene Expression—RT-PCR Analysis.

At week 2 post stent placement, the stented femoral arteries (n=3stents/group) were harvested from Coated (+) and Coated (−) groups anddivided laterally into three sections (proximal, mid and distal) afterremoving the stents. A part of the sections were used to detect hVegftranscript by RT-PCR, while the remaining parts were used for to detectthe hVegf protein by immunostaining and reendothelization by staining.Similarly, sections were also collected at week 16. For detection ofVegf gene expression, total RNA was extracted from stented arterysamples (stored in RNAlater™, Qiagen) using RNA Extraction kit (Qiagen)according to the manufacturer's instructions. The obtained RNA wasquantified and reverse transcribed to Vegf cDNA using the Qiagen'sReverse Transcription (RT) kit following the supplied instructions. PCRamplification was performed on the reverse transcribed product using TaqDNA Polymerase (Invitrogen) and forward primer(5′CTTGCCTTGCTGCTCTACCTCC3′) (SEQ ID NO: 1) and reverse primer(5′GCTGCGCTGATAGACATCCATG3′) (SEQ ID NO: 2) for hVegf gene (112-bpproduct). Amplifications were carried out for 25 cycles at 94° C. for 35s (denaturation), 57° C. for 35 s (annealing), and 72° C. for 25 s(extension).

Immunohistochemical Studies.

For immunostaining, paraffin embedded 5 μm sections were deparaffinised,blocked with donkey serum and incubated over night with 1:50 dilution ofrabbit anti-hVegf (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.)primary antibodies. On the second day, the slides were thoroughly washedwith wash buffer and incubated with donkey anti-rabbit IgG-FITC (SantaCruz Biotechnology Inc.) with 1:200 dilutions for 1 hour. Theproportions and intensities of FITC-positive regions in the tissuesections, as seen under fluorescence microscope (Nikon EclipseTE2000-U), gave a qualitative idea of the relative amount of hVegfexpressed in the stented vascular tissue regions due to the transgenedelivery from stent platform.

Assessment of Stent Re-Endothelization—Evans Blue Staining.

Two weeks after stent deployment animals were anesthetized (n=3, Coated+and −) and a portion of the harvested artery was incubated in 1% Evansblue (Sigma-Aldrich, Dublin, Ireland) for 15 min, followed by washing,fixing and examining longitudinally using image software as mentionedelsewhere (Sharif F et al. 2008).

Scanning Electron Microscope.

Re-endothelialization was also assessed at 2 week and 16 week postimplantation (n=3 each). Retrieved stents were washed with saline, fixedin 4% paraformaldehyde and longitudinally incised as above and examinedusing scanning electron microscopy (SEM).

Histological Assessment.

Stents were retrieved after twelve weeks and harvested vessels wereembedded in methylmethacrylate plastic (Accel Lab Inc, Quebec, Canadaand McGill SAIL Lab). After polymerization, the proximal, mid and distalsections of the stents were cut into 5 μm sections and stained withhematoxylin and eosin. Re-endothelialization was assessed directly underthe microscope by a histologist blinded to treatment. Endothelialcoverage was expressed as the percentage of the average lumencircumference covered by the endothelial cells (Sharif F et al. 2008).

Assessment of ISR—Angiogram Analysis.

Initial and follow-up angiograms were performed with fluoroscopicangiography (GE Stenoscop) using Omnipaque Iohexol contrast dye inanterior oblique projection and percent diameter stenosis at follow-upwas calculated by (minimal stent diameter at follow-up/the mean diameterof the stent at full expansion)×100, using standard procedure asdescribed elsewhere (Kornowski R et al. 1998). Sections were also usedto evaluate the presence of inflammation and vessel wall injury at thestented sites of all the groups by injury and inflammation score method(Schwartz R S et al. 1992).

Morphometric Analysis.

Retrieved vessels were embedded in methylmethacrylate plastic, cut intothin sections as mentioned earlier, and stained with elastic Van Giesonstain. All sections were examined by light microscopy and photographedto quantify the neointima formation and stenotic area using methodsmentioned elsewhere (Schwartz R S et al. 1992; Schwartz R S et al.1990).

Statistical Analysis.

Quantitative variables are presented as mean±Standard Deviation (SD)from independent experiments as described in the figure legends.Statistics were performed using student's t-test or one-way ANOVA byBonferroni's multiple comparison post-hoc test. All statistical analyseswere performed with Prism 5 (GraphPad Software). P value<0.05 wasconsidered significant.

Example II In Vitro and In Vivo Characterization of Embedded BaculovirusEncapsulation of Baculovirus in PLGA Microspheres.

In order to encapsulate the baculoviruses, the method of preparation andenergy source required for formation of the primary emulsion werestudied extensively and optimized in vitro. Bac was added to PLGAdissolved in DCM and the mixture was either sonicated or homogenized inpresence or absence of BSA/glycerol. The viral titer was checked beforeand after encapsulation in order to measure the encapsulationefficiency. As the viral titer measures only the amount of active virus,the data represented as the amount of encapsulated virus denotes theactual number of active virus, and not the overall number which willconsist of both active and inactive/disrupted viral particles. Our data,as presented in Table 1, reveals that sonication method did not increasethe encapsulation efficiency; in fact, it has a deleterious effect onthe viral viability compared to mechanical homogenization technique. Onthe other hand, homogenization technique produces the water oil emulsionin a much simpler mechanical agitation method which resulted in lesserloss of active virus. To further improve the encapsulation method,BSA/glycerol was mixed with the viruses to increase the virus stability,to enhance their steric hindrance against the harsh external shearstress during MS preparation and for better cryopreservation.

TABLE 1 Effect of PLGA MS preparation procedure on active virusencapsulation efficiency (in terms of percentage of initial loaded viraltitre) Homogenization Sonication Bac + Bac + Method Bac BSA/glycerol BacBSA/glycerol w/o/w (% pfu) 15.5 ± 0.7 35.6 ± 3.1 10.6 ± 1.3 26.4 ± 4.1Diameter (μm)  7.2 ± 2.3  9.8 ± 3.4  5.2 ± 3.4  5.6 ± 4.2 w/o/o (% pfu)21.0 ± 1.1 41.4 ± 2.2 17.8 ± 1.0 26.8 ± 3.5 Diameter (μm)  9.2 ± 2.6 8.3 ± 3.2  5.6 ± 1.1  4.9 ± 2.2

It was then determined if the second emulsion solvent or the continuousphase can affect the encapsulation efficiency. An hydrophobic (corn oil)solvent was used for second emulsion step and compared it with standardhydrophilic (PVA) solvent. Oil was selected instead of water in order toreduce the viral loss in the second emulsion phase and subsequentsolvent evaporation step. A significantly higher amount of encapsulationefficiency was noticed in the w/o/o method which confirms that there wasless viral loss using oil compared to water in the continuous phase. Theparticle diameters were checked for every preparation which variedbetween 5-10 μm. Thus, as evident from Table 1, a w/o/o double emulsionprocedure using mechanical homogenizer can efficiently encapsulate thebaculoviruses which can be further enhanced by supplementing them withBSA/glycerol to protect the viral activity.

SEM photomicrographs were taken to confirm the formation of sphericalshaped virus loaded microparticles (FIGS. 2A and 2A′), while the AFMimages (FIGS. 2B i and 2B ii) identifies the regular, uniform andconsistent surface topography of the microspheres. TEM imagesdemonstrate empty and baculovirus loaded microspheres, while FIG. 2Ereconfirms the successful microencapsulation procedure showing theentrapped baculoviruses in the cut section of a virus loaded PLGAmicrosphere.

Baculovirus Eluting Stent: Physical Characteristics and In Vitro ReleaseProfile.

The prepared polymer coated stents containing the PLGA microspheres (byw/o/o method), mixed with Red Nile dye as tracer, were air dried andphotographed to confirm that the polymeric stent was able to hold theembedded microspheres after stent expansion. FIGS. 3A and 3B shows astent before and after coating with MS embedded fibrin layers, while thepinkish color of the stent in FIG. 3C confirms retention of the loadedMS on the stent surface post stent expansion using balloon catheter. Afluorescence image of the coated stents under fluorescence microscope(Nikon Eclipse TE2000-U) as shown in the FIG. 3D further confirms theabove findings, where a uniform coating of the microspheres was seen onthe stent surface. SEM images of the stents (FIGS. 3E to 3G) providedfurther morphological evidences on the surface characteristics of thecoated stents demonstrating the uncoated bare metal stent struts (FIG.3E), plain surface of the uniformly coated stent struts with fibrinmultilayers (FIG. 3F) and evenly distributed PLGA MS embedded on thefibrin multilayered stent strut surfaces.

To determine the release kinetics of the encapsulated baculoviruses fromthe stent surface and modulate their release behavior, two differentPLGA concentrations (50 and 100 mg/ml of DCM) were used using w/o/oemulsion method and compared with standard w/o/w method.

In both the methods the stent group with MS prepared from lower PLGAconcentration showed much faster and higher percentage of viral releasewith time compared to the higher concentration (FIG. 3H). Moreover,w/o/o group showed significantly higher release of active virus at lowerPLGA concentration than w/o/w group, 24 h post-incubation in PBS. Afurther decrease in PLGA concentration led to non-uniform MS formationsand hence a concentration of 50 mg/ml DCM PLGA MS in the formulationprotocol using w/o/o method for further studies.

Baculovirus Surface Functionalization with PAMAM Dendrimer EnhancesTransduction.

To confirm the successful formation of Bac-PAMAM complex, zeta potentialof the microparticles were measured as shown in FIG. 4A. At pH 7.4, thezeta potential of the free baculoviruses was negatively charged with azeta potential of −13.4 mV±0.6. The positively charged PAMAM dendrimers,upon conjugation with the negatively charged baculoviruses, formedmicroparticles which resulted in an increment of surface charge towardspositive. Bac-PAMAM (0.01) showed a zeta potential of −10.6±1.8 mv,while Bac-PAMAM (0.1) showed −2.36±2.2 mv, Bac-PAMAM (0.05) showed8.7±2.4 mv and Bac-PAMAM (1.0) showed 11.5±4.7 mv, where the valueswithin brackets indicate the ratio of PAMAM/virus used in preparing themicroparticles. This was further confirmed by measuring the particlesize of the formed microparticles (FIG. 4B). Free baculovirus showed anaverage size of 226±17.6 nm, while Bac-PAMAM (0.01) showed a size of228.5±30.8 nm, Bac-PAMAM (0.1) showed 588±54.1 nm, Bac-PAMAM (0.05)showed 328±29.5 nm and Bac-PAMAM (1.0) showed 289±27 nm. Thesignificantly increased size of the Bac-PAMAM (0.05) particles comparedto the free baculovirus indicates the efficient formation of Bac-PAMAMmicroparticles, generated by strong electrostatic interactions betweenbaculovirus and PAMAM dendrimers. The excessively bigger size inBac-PAMAM (0.1) group is probably because of the formation of largeraggregates and clumps of the formed complexes. To reconfirm the complexformation, TEM images the products were taken for morphologicalevidences. FIGS. 4C and D demonstrate the efficient binding of the rodlike baculovirus with PAMAM dendrimers (0.05) leading to the formationof stable Bac-PAMAM hybrid complex.

Baculovirus-PAMAM Eluting Stent: In Vitro Release, Transduction andCytotoxicity Analysis.

To investigate whether the released BacMGFP were bioactive and cantransduce the HASMCs, the incubation buffers from different release timepoints were collected, diluted in same proportions and added to HASMCsfor transduction. After 72 h, the GFP expressions were quantified interms of percentage cells transduced. For the study, four differentbaculovirus formulations using varied concentrations of PAMAM dendrimerswere prepared before encapsulating and loading them on the stentsurface. The data in FIGS. 5A to 5E demonstrates that PAMAM dendrimerfunctionalization has a positive effect on baculovirus mediatedtransduction with BacMGFP-PAMAM (0.5) showing consistently highertransduction efficiency than other groups, including free BacMGFP(control). Further increasing the PAMAM concentration in BacMGFP-PAMAM(1.0) however did not show any significant improvement. Moreover, thepercentage of transduction was proportional to hour of incubationbuffer. This is because of the higher amount of baculovirus released inthe incubation buffer with higher incubation time, although there wereno significant differences in transduction efficiencies between 18 h and24 h in all groups. This indicates that the active viruses were mostlyreleased within 12 h to 18 h from the stent. Furthermore, 72 hcytotoxicity studies on the effect of these different formulations onHASMCs show that BacMGFP-PAMAM can be safely used for cell transduction,although BacMGFP-PAMAM (1.0) group with high PAMAM concentration showedsignificantly high cytotoxicity compared to the control (FIG. 5F).

Effect of Serum and Storage Condition on Bioactivity of the Stent.

The in vivo applications of baculovirus are limited mostly because oftheir serum inactivation. The stents were incubated in 50% FBS for 1 hfollowed by incubation in PBS for 24 h. As control, 50% FBS was replacedby PBS solution. The incubation buffer was then used to transduce theHASMCs as mentioned earlier and GFP expression was detected andquantified using plate reader. FIG. 5A shows that compared to controlPBS, serum has an inactivation effect in all the groups which was moredistinct BacMGFP stent control group where baculoviruses were loadeddirectly on the stent surface without microencapsulation. In the BacMGFPMS stent group, there was much higher GFP expression compared to Bacstent in presence of serum demonstrating that the MSs were able toprotect the entrapped baculoviruses against serum inactivation. However,BacMGFP-PAMAM MS stent group showed highest GFP expression and hence,highest protection against serum. This may be because of the combinedeffect of the MS encapsulation and PAMAM coating on the viral surface.

To further understand whether the prepared stents can be stored underdifferent storage conditions, they were stored under differenttemperature conditions for long term storage of 3 months. After thawingand incubation in PBS solution, the incubation buffers were used totransduce the HASMCs as described earlier. FIG. 5B demonstrates that−80° C. is ideal for long term storage compared to storage at 4° C. and−20° C., although there was significant difference in GFP expressionwhen these groups were compared to freshly prepared one.

Quantification and Functional Analysis of Expressed hVegf fromTransduced HASMCs.

Conditioned media, collected from BacVegf-PAMAM (0.5) and BacVegftransduced HASMCs at regular intervals, were used to quantify and detectthe Vegf release profile using a hVegf ELISA. CM from non-transducedcells was taken as the negative control. The data, as shown in FIG. 7A,demonstrates rapid expressions of hVegf in BacVegf-PAMAM and BacVegfgroups in the first 4 days which gradually decreased over the week.Although Vegf expression decreased considerably in BacVegf group afterthree weeks, BacVegf-PAMAM group maintained significantly higher Vegfexpression.

The bioactivities of the released hVEGF in media from the BacVEGF-PAMAM(0.5) transduced HASMCs were evaluated in vitro by observing theproliferative capacity of the HUVECs. Cell Proliferation MTS Assay kitwas used to assess the proliferation capabilities of the HUVECs treatedwith CM (containing secreted hVEGF) from experimental samples, collectedon day 4 post transduction. As shown in FIG. 7B, groups BacVegf andBacVegf-PAMAM showed significantly high HUVEC proliferation on day 3compared to unstimulated control, with BacVegf-PAMAM exhibiting thehighest proliferation (3.98±0.19×10⁴ cells). As expected, group treatedwith antibodies against hVEGF showed no proliferative effects provingthat it was because of the bioactivity of released hVEGF fromgenetically modified SMCs that contributed to the drastic HUVECproliferations. This proliferation rate was directly dependant on theamount of the VEGF released which explains why BacVegf-PAMAMdemonstrated better results than BacVegf and unstimulated controlgroups.

Similar results were noticed in the wound healing assay where theabilities of CM from different experimental groups, collected on day 4post transduction, to promote HUVEC migrations were measured. Asdepicted in FIG. 7C, stimulation of wounded HUVEC monolayer with CM fromBacVegf-PAMAM exhibited significant healing of wounded area (89.4±7.5%)compared to CM from unstimulated control (12.4±2.4%) and BacVegf(61.7±5.5%). Pre-incubation of CM with the neutralizing anti-VEGFantibodies completely hindered BacVegf-PAMAM CM induced wound healing,clearly suggesting that chemotactic signals from hVEGF are required forproper wound healing effect.

In addition, the biologic activities of the CM were also evaluated byHUVEC tube formation assay. As illustrated in FIG. 6Di, cells treatedwith CM from BacVegf-PAMAM induced significantly enhanced effect onHUVEC capillary network formation as compared to the cells treated withCM from BacVegf and unstimulated groups. The relative tubule length wassignificantly enhanced in BacVegf-PAMAM and BacVegf group compared tocontrol (141.5±7.9 and 100±13.6 vs. 47±2.1). Also, to assess the extentof impact of hVegf present in the CM on HUVEC tube formation, anti-VEGFantibody was added to the supernatant with released CM. A significantlyreduced tube formation was observed which provides evidence of thestrong pro-angiogenic nature of VEGF present in the CM. Similar resultswere also noticed when total capillary tubule numbers were quantified inBacVegf-PAMAM and BacVegf group and compared to unstimulated control(15.5±3.4 and 10.1±2.2 vs. 4.5±0.7) in FIG. 6Dii.

Detection and localization of Vegf expression in vivo. The in vivo stentimplantation procedure has been demonstrated in FIG. 8A, where the dogsunderwent bilateral femoral artery denudation by balloon angioplasty(FIGS. 8Ai and i), followed by stent deployment at the injured site(FIG. 8Aiii). Stented femoral arteries transduced with Coated (+) andCoated (−) were harvested 14 days after stent placement. Followingqualitatively reverse transcriptase-PCR analysis, hVegf signalexpression could be observed in all the proximal, middle and distalsections of the BacVegf-PAMAM (Coated+) transduced arteries examined(n=3 vessels), while nothing was detected in BacNull-PAMAM (Coated-)transduced arteries (FIG. 8B). Although, the RT-PCR product from 2 weeksBacVegf-transduced tissue samples demonstrated the presence of theappropriate-sized band for hVegf in the stented artery sections, nobands were detected from 16 weeks samples proving that the transgeneexpression is transient and disappears over time. Moreover, arterysections from 1 cm proximal and distal to the stented artery did notshow any transgene expression, thus indicating that the gene deliverywas localized and restricted to the stented region where the viruscontaining polymer coated strut touches the inner wall of the artery.

Stent-based delivery of BacVegf to the canine femoral arteries resultedin localized overexpression of Vegf. This transgene expression waslocalized to the areas around the stent struts in the intimal and mediallayers as demonstrated elsewhere with β-galactosidase gene deliveryusing adenovirus and adeno-associated virus-coated stents (Sharif F etal. 2006). The expression detected on day 14 was localized near thestent strut region, mainly on the medial side where the polymer of thestent touches the arterial surface; in contrast, there was no Vegfstaining observed in the stented arteries treated with Coated (−)stents.

Endothelial Recovery in Stented Arteries.

Endothelial regeneration was determined using three independentmethodologies: Evan's blue staining, scanning electron microscopy andhistology. Two weeks after balloon injury and stent placement,endothelial regeneration was assessed in the animals treated withBacVegf-PAMAM and BacNull-PAMAM coated stents using Evans blue staining(n=3 stent). Luminal staining for Evans blue demonstrated that theballoon angioplasty and stenting procedure completely denuded thefemoral artery in Control (−) stent while Coated (+) showed markedrecovery at week 2 (FIG. 9A). As evident from FIG. 9B,re-endothelialization was significantly greater in the Coated (+)vessels (55.36±4.64%) in comparison with Coated (−) control vessels(37.5±6.51%, P<0.05).

Furthermore, at 2 weeks and 16 weeks after stent deployment, SEMpictures of the inner surface of the stented arteries were taken. Cellsconsistent with endothelial morphology were noted on the surface ofBacVegf-treated stent struts on week 2, which completely covered thestent surface in a uniform way after 16 weeks (FIG. 9C). In contrast, anirregular rough surface, mainly comprising of the exposed stent strutand partially covered neointima tissues, was noticed in the arterieswith BacVegf-treated stent on both week 2 and week 16.

Sixteen (16) weeks after the stent placement, histological assessment ofendothelial regeneration demonstrated a significant difference inendothelial regeneration between the three groups [Coated+, Coated- anduncoated bare metal stents]. The percentage of endothelial cellsobserved in the Coated (+) vessels was significantly higher than in thecontrol vessels (93.5±5.2% vs. 75.4±9.3% and 72.4±4.1; P<0.05) as shownin FIG. 9D.

Detection of Stenotic Area in Stented Artery: Morphometric andAngiographic Analysis.

All vessels were angiographically and histologically patent throughoutthe period of study. Stent malapposition was also not detected in anyanimal. 16 weeks after site-specific Vegf gene transfer, the stenoticarea was significantly reduced in Coated (+) compared to control groupstents (54.58±14.1% vs. 69.6±15.51 vs. 85.4±10.14%; P<0.05) as analyzedby angiography (FIG. 10). Representative photomicrographs of histologiccross sections from stented arterial segments at 4 month follow up areshown in FIG. 10. The extent of vessel injury at the stent site wassimilar in all the groups as determined by injury score (Coated (+)1.13±0.14 vs. Coated (−) 1.15±0.27 vs. Uncoated 1.21±0.35). Similarresults were also obtained with inflammation score (Coated (+) 0.66±0.34vs. Coated (−) 0.7±0.31 and Uncoated 0.81±0.45; P>0.05).

Histomorphometric analysis (FIG. 11) demonstrated a significantlyreduced intimal hyperplasia in Coated (+) group compared to other twogroups (FIG. 11E, 61.36±15.15% vs. 78.41±13.84 and 87.66±8.54%; P<0.05).Similar results were obtained when analyzed in terms of cross-sectionalmean neointimal area (FIG. 11E, 2.23±0.56 mm² vs. 2.78±0.49 mm² and3.11±0.23 mm²; P<0.05). One (1) cm proximal and distal to the stentedarea showed no signs of intima formation.

ISR caused by intimal hyperplasia post angioplasty and stenting remainsa major cause of concern and challenge for clinical investigators andresearchers. To develop a therapeutic strategy which would reduce ISR byaccelerating local re-endothelization at the stented site, a genetherapy was selected as it offers a promising tool for the treatment ofISR. Efficient gene therapy using suitable nanodelivery systems caninduce a therapeutic effect for several days, whereas the half-life ofrecombinant protein and other pharmaceutics in circulation is muchlimited and proved to be ineffective in certain human. Although previouspreclinical studies have shown promising results and clinical studieshave demonstrated the safety and feasibility of vascular gene transferin patients, none of the randomized controlled phase-II/III gene therapytrials have shown relevant positive effects on inhibiting ISR. Thereasons may be inadequate dose response effect due to low gene transferefficacy, cell-mediated immunity to virus-transduced cells, lack ofsophisticated vector delivery method or an amalgamation of severalinterrelated factors. Taking into account the technical andpharmacological shortcomings of previous studies, it was determined todevelop a clinically relevant gene eluting stent that can successfullyameliorate the vascular biology of the stented site by promoting localendothelial recovery. Novel dendrimer coated BacVegf eluting stent weredesigned and developed using a composite polymer containing PLGAmicrospheres and fibrin biopolymer as the gene delivery platform. Theresults demonstrated here confirm that Vegf transgene can be effectivelydelivered to the vessel wall by the microencapsulated baculovirus, asdetected in vivo in the transcript and protein level on day 10 poststenting. The transgene delivery not only improved earlyreendothelialization at the stented site but also significantly reducedneointimal proliferation as assessed by angiography andhistomorphometric data at 4 months of follow-up. Prior studies havedemonstrated that 4 month study period can be considered as anappropriate end point to detect vascular responses post stenting, afterwhich the intimal response stabilizes with little change over the next 2years.

In this work, baculoviruses were microencapsulated using a new procedureof w/o/o double emulsion to restrict viral loss to the hydrophilic phaseduring second emulsion, achieve high encapsulation efficiency and obtainfavorable release profile of the active baculoviruses. This methodproved much effective in maintaining higher encapsulation efficiencycompared to earlier used w/o/w method. Baculovirus formulated in PLGAmicrospheres was released in two phases, where an initial burst releasewas noticed within 12 h of incubation followed by a slow, continuousrelease in next 12 h where the PLGA microspheres undergo slowdegradation by hydrolysis of ester linkages to yield lactic and glycolicacid. Both mechanical homegenization and sonication inducedemulsification procedures were used to entrap the viruses within themicrospheres. But, sonication process reduced the encapsulationefficiency of active baculoviruses compared to that prepared byhomogenization.

Recently, potential of recombinant baculovirus for angiogenic therapy inmyocardially infarcted animal model using a nanobiohybrid system wasreported (Paul A. et al. 2011). To extend its prospect for furtherangiogenic therapy applications, improvement of transduction efficiencywas made by hybridizing the baculovirus with cationic hyperbranchedPAMAM G0 dendrimers before microencapsulating them. The amino terminalgroups of dendrimer coated baculovirus could facilitate theelectrostatic interactions with negatively charged cell surface whichaugment viral binding to the cell surface and their subsequent entryinto the cells. Our present study demonstrates that surface modifyingthe baculovirus with positively charged PAMAM dendrimers can alsoimprove gene transfer efficiency. Among other widely used polymers,polyamidoamine (PAMAM) dendrimers has been shown to function as highlyefficient cationic polymer vectors for gene delivery. PAMAM dendrimersare synthetic nanoparticles with a unique molecular architecture,characterized by their well defined structure, high degree ofinter-molecular uniformity, low degree of polydispersity, and multipleterminal amino functional groups. Most importantly, it can be anexcellent polymer for conjugation of functional molecules whilemaintaining low toxicity in vitro and in vivo.

Aside from improved transduction, the present work demonstrates thefeasibility of encapsulating dendrimer-coated active recombinantbaculovirus into microspheres in order to achieve a controlled releasefrom polymeric stent surface with minimum cytotoxicity. Veryimportantly, the microencapsulated Bac-PAMAM stent also showedsignificantly higher protection of bioactive baculoviruses against seruminactivation compared to stents with microencapsulated Bac, andnon-encapsulated Bac and Bac-PAMAM stents. Our data confirms that evenPAMAM coating can also protect the Bac against serum inactivation tosome extent, which is further enhanced by encapsulating them inside PLGAmicrospheres. The preservation of bioactivity of the stent for at least3 months, once stored at −80° C., can be of significant logisticadvantage under real life clinical settings, where the stored stent canbe of immediate off-the-shelf use for any patient undergoing angioplastyand stenting without delay.

As a first step to evaluate the therapeutic potential of this newlyformulated gene eluting stent, stent were loaded with Vegf carryingbaculovirus and in vitro analysis were performed. So far drug-elutingstents has proved to be a useful strategy for the prevention of ISRusing antiproliferative drugs like rapamycin, paclitaxel and everolimus.But recent clinical outcomes indicate that this approach is leading toincomplete endothelization and associated risk for stent thrombosis. Theunderpinning cause for this is that the drugs, apart from restrictingthe smooth muscle proliferation, impinge the natural endothelialregeneration process. Thus improving the regenerative capacity of thevessel wall endothelial cells, impaired by the antiproliferative agents,is critical to alleviate the risk of stent thrombosis and neointimaformation. Earlier studies have shown that site specific arterial Vegfgene transfer lead to collateral vessel formation and increasedcapillary density in ischemic tissues. As a follow up work, severalstudies reported that exogenous balloon and stent delivered Vegf genehas the potential to accelerate thrombus recanalization and promotere-endothelization in denuded arteries. This, in turn, attenuates ISRand inhibits stent thrombosis. Our in vitro and in vivo findings are insync with these prior findings and further focuses on advancing the genedelivery technology using optimized stent/polymer/gene combination.Here, it is illustrated that the viruses released from the microsphereembedded stent can efficiently transduce the HASMCs in culture, withBacVegf-PAMAM showing significantly higher expression of Vegf comparedto that with BacVegf. In addition, in vitro functional assessment byHUVEC proliferation, wound healing and tube formation assays confirm thebiological potency of the expressed Vegf protein.

Preclinical studies have confirmed the safety and efficacy of fibrincoated stents post deployment. With that knowledge, the development abiocompatible stent surface by coating the metallic stent with layers offibrin hydrogel, impregnated with baculovirus loaded biodegradable PLGAmicrospheres was sought. The topmost genipin/fibrin layer serves as thebarrier to external damages during crimping on the balloon catheter aswell as protects the inner layers from premature virus release duringits passage through the lumen of the artery at time of implantation atthe desired site. The addition of genipin, as a natural cross-linker tofibrin is believed to work towards reducing the chance of anyinflammatory reactions. Through the stent coating method presented here,the inner surface of the stent (i.e., the mandrel contact surface) wasnot coated with the polymer. Consequently, there was no fibrin coatingon the blood contact side of the sten to reduce the loss of loadedviruses into the blood stream.

As illustrated in this study, the stent acted as an ideal platform forlocal baculovirus delivery to the stented blood vessel wall with nosigns of potential inflammatory responses in the artery. Notably, therecombinant baculovirus showed a rapid expression of transgene withinthe first 4 days of in vitro stent mediated transduction, followed by agradual decrease in expression level over the next two weeks. Similarly,it was observed localized in vivo Vegf transgene expression at the siteof stent implantation for at least 2 weeks post deployment as evident byRT-PCR and by immunohistochemical analysis, where the later confirmedgene over-expression around the stent struts. As expected, theexpression ceased when analyzed at the transcript level on week 16. Thistransient nature of baculovirus expression indicates that it can beadvantageous in treating problems like ISR where the gene expression isno longer needed once endothelial recovery is complete. The present workproves this postulation showing complete recovery of endothelial layeris possible by temporal expression of transgene for 2-3 weeks. Thisobservation supports previous published results, where transgeneexpression for just 10 days proved sufficient for complete endothelialrecovery at the stented site. More importantly, this transient nature ofbaculovirus expression makes it a prospective gene delivery vehicle forbiologically safer clinical applications compared to widely experimentedmammalian viral systems.

While the disclosure has been described in connection with specificembodiments thereof, it will be understood that the scope of the claimsshould not be limited by the preferred embodiments set forth in theexamples, but should be given the broadest interpretation consistentwith the description as a whole.

REFERENCES

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1. A viral microparticle for the delivery of a recombinant therapeuticnucleic acid molecule to the cells of a vertebrate individual, saidviral microparticle comprising: (i) a matrix of biocompatiblebiodegradable polymers; and (ii) a genetically-engineered baculovirushaving a viral genome comprising the recombinant therapeutic nucleicacid molecule, wherein the genetically-engineered baculovirus cannotreplicate in the cells of the vertebrate individual.
 2. The viralmicroparticle of claim 1, wherein: (a) the biocompatible biodegradablepolymer comprises a polyester; or (b) the viral microparticle of (a),wherein the polyester comprises a poly(lactic-co-glycolic acid). 3.(canceled)
 4. The viral microparticle of claim 1, wherein thegenetically-engineered baculovirus: (a) is from the generanucleopolyhedrovirus; (b) is a multicapsid virus; (c) is from thesubtype Autographa californica multicapsid nucleopolyhedrovirus(AcMNPV); or (d) is at least partially embedded in the matrix. 5.-7.(canceled)
 8. The viral microparticle of claim 1, wherein: (a) therecombinant nucleic acid molecule encodes a growth factor; (b) the viralmicroparticle of (a), wherein the growth factor is an angiogenic growthfactor; or (c) the viral microparticle of (b), wherein the angiogenicgrowth factor is vascular endothelial growth factor (VEGF). 9.-10.(canceled)
 11. The viral microparticle of claim 1: (a) furthercomprising a coat of cationic polymers covering at least a portion ofthe surface of the baculovirus; (b) the viral microparticle of (a),wherein the cationic polymers are dendrimers; (c) the viralmicroparticle of (b), wherein the dendrimers are poly(amidoamine)(PAMAM); or (d) the viral microparticle of (c), wherein the PAMAM is aG0 PAMAM. 12-14. (canceled)
 15. The viral microparticle of claim 1: (a)wherein the viral microparticle has a relative size of at least 5 μm; or(b) wherein the viral microparticle has a relative size a relative sizeequal to or less than 10 μm.
 16. (canceled)
 17. A process for making aviral microparticle for the delivery of a recombinant therapeuticnucleic acid molecule to the cells of a vertebrate individual, saidprocess comprising: (a) resuspending, in an aqueous solution, agenetically-modified baculovirus having a viral genome comprising therecombinant therapeutic nucleic acid molecule so as to obtain an aqueouspreparation; (b) combining and homogenizing the aqueous preparation ofstep (a) with a solution of a biodegradable biocompatible polymer and awater immiscible, volatile organic solvent so as to form a water-in-oil(w/o) emulsion; (c) combining and homogenizing the water-in-oil emulsionof step (b) with an oil so as to form a water-in-oil-in-oil (w/o/o)emulsion; and (d) evaporating the water immiscible, volatile organicsolvent from the water-in-oil-in-oil emulsion of step (c) so as to formthe viral microparticle.
 18. The process of claim 17, further comprising(e) recuperating and washing the viral microparticles obtained in step(d).
 19. The process of claim 17, wherein: (a) the oil further comprisesa surfactant; (b) the process of (a), wherein the surfactant is anon-ionic surfactant; (c) the process of (a), wherein the oil is avegetable oil. 20-21. (canceled)
 22. The process claim 17, furthercomprising, prior to step (a), coating, at least partially, thegenetically-modified baculovirus with a cationic polymer.
 23. A viralmicroparticle for the delivery of a recombinant therapeutic nucleic acidmolecule to a vertebrate individual obtained by the process claim 17.24. A pharmaceutical composition comprising: (a) (i) a gelling agent and(ii) the viral microparticle of claim 1; (b) the pharmaceuticalcomposition of (a), wherein the gelling agent comprises a protein and/ora protein fragment; (c) the pharmaceutical composition of (a), whereinthe gelling agent comprises a fibrinogen and/or a fibrin. 25-26.(canceled)
 27. A process for formulating the pharmaceutical compositionof claim 25, said process: (a) comprising combining the gelling agentand the viral microparticle; or (b) further comprising cross-linking thegelling agent.
 28. (canceled)
 29. A kit for making the pharmaceuticalcomposition of claim 25, said kit comprising: (a) (i) the gelling agentand (ii) the viral microparticle; or (b) (i) the gelling agent, (ii) theviral microparticle and (iii) a cross-linking agent.
 30. (canceled) 31.A support comprising the viral microparticle of claim
 1. 32. A supportcomprising: (a) the pharmaceutical composition of claim 24; (b) thesupport of (a), wherein the support is a solid or comprises a solidmaterial; (c) the support of (a), wherein the support is metallic orcomprises a metallic material; (d) the support of any of (a) to (c),being or manufactured as a medical implant; or (e) the support of (d),wherein the medical implant is a stent. 33-36. (canceled)
 37. A methodof promoting the healing of a wound in a vertebrate individual in needthereof, said method comprising placing a therapeutically effectiveamount of the viral microparticle of claim 1, in the vicinity of thewound, thereby promoting the healing of the wound in the vertebrateindividual.
 38. A method of promoting the healing of a wound in avertebrate individual in need thereof, said method comprising placing atherapeutically effective amount of viral microparticle obtained by theprocess of claim 17 in the vicinity of the wound, thereby promoting thehealing of the wound in the vertebrate individual.
 39. A method ofpromoting the healing of a wound in a vertebrate individual in needthereof, said method comprising placing a pharmaceutical composition ofclaim 24 in the vicinity of the wound, thereby promoting the healing ofthe wound in the vertebrate individual.
 40. A method of promoting thehealing of a wound in a vertebrate individual in need thereof, saidmethod comprising placing a pharmaceutical composition obtained by theprocess of claim 27 in the vicinity of the wound, thereby promoting thehealing of the wound in the vertebrate individual.
 41. A method ofpromoting the healing of a wound in a vertebrate individual in needthereof, said method comprising placing a support of claim 32 in thevicinity of the wound, thereby promoting the healing of the wound in thevertebrate individual.